Apparatus and method for providing assay calibration data

ABSTRACT

A semi-automated biological sample analyzer and subsystems are provided to simultaneously perform a plurality of enzyme immuno assays for human IgE class antibodies specific to a panel of preselected allergens in each of a plurality of biological samples. A carousel is provided to position and hold a plurality of reaction cartridges. Each reaction cartridge includes a plurality of isolated test sites formed in a two dimensional array in a solid phase binding layer contained within a reaction well which is adapted to contain a biological sample to be assayed. The carousel and cartridges contain structures which cooperate to precisely position the cartridges in each of three separate dimensions so that each cartridge is positioned uniformly. An optical reader operating on a principle of diffuse reflectance is provided to read the results of the assays from each test site of each cartridge. Also provided is a subsystem which provides predetermined lot-specific assay calibration data which is useful for normalizing the results of various assays with respect to predetermined common standard values.

This application is a continuation of application Ser. No. 227,586,filed Aug. 2, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to biological sample analyzersand more specifically to a semi-automated analyzer and subsystemsthereof capable of simultaneously carrying out a panel of assays on eachof a plurality of different biological samples. In one aspect, theanalyzer of the invention is adapted to simultaneously assay each of aplurality of biological fluid samples for human IgE class antibodiesspecific to a preselected panel of allergens.

A significant portion of the population has some allergic reaction tosubstances such as pollen, animal dander, or other commonly presentallergenic substances. A key element in the treatment of such allergicsymptoms is identification of the particular substance to which a personmay be allergic. Previous methods for determining allergichypersensitivity were performed using direct skin tests on the patient.In these direct skin tests minute quantities of various allergens wereinjected into or under the patient's skin and the particular patch ofskin was subsequently examined to determine whether or not a person hadan allergic reaction to the previously introduced allergen.

In addition to being uncomfortable for the patient, patients on certainmedications (i.e. antihistamines) cannot be accurately tested by directskin tests.

Accordingly, a number of in-vitro testing procedures have beendeveloped. Such procedures detect circulating IgE in serum or plasma orother microbiological interreactions using an insoluble solid carriercoated with a known quantity of antigen extract derived from a knownallergenic substance. The coated carrier is typically exposed to, andincubated in, a sample of the patient's blood serum. If the patientcarries the IgE class antibody which is specific to the particularallergen and which is the cause of the patient's allergic reaction tothe allergen, a measurable binding reaction occurs on the carrier duringthe incubation period. The concentration of the IgE antibody in thesample and accordingly the degree of allergic sensitivity of the patientis then determined by measuring the magnitude of the binding reactioneither visually, photometrically, fluorometrically, radiologically,enzymatically, or by other known techniques.

While such in-vitro procedures provide advantages over in-vivo testingprocedures, they are not without disadvantages. First, a relativelylarge quantity of blood is required to test the patient's sensitivity toa large number of specific allergens. Second, testing for a large numberof different allergens in separate cuvettes is tedious and timeconsuming for the physician or technician performing the test.

To this end, efforts have been devoted to develop a system whichsimultaneously tests for a number of specific allergens utilizing asingle sample of the patient's blood serum. For example, U.S. Pat. Nos.3,941,876 (Marinkovich) and 4,031,197 (Marinkovich) disclose techniquesfor the screening of different IgE class antibodies. The techniquestaught by Marinkovich involve coating an elongated cellulosic body, suchas a strip of paper, with separate identified allergens to form bands orislands, which are separated from one another by allergen-free areas.The coated cellulose material is then contacted with a test serum sothat serum IgE class antibodies specific for the coated allergens willbind to the appropriate bands or islands. The cellulosic body is thenwashed and subsequently incubated with labelled antibodies that arereactive with the attached IgE class antibodies. The bands or islandsare then analyzed for the presence of the labeled antibodies.

U.S. Pat. No. 4,459,360 (Marinkovich) also discloses a similarmultiple-component binding assay system which includes a plurality ofcoated filaments mounted on a support for simultaneously screening aliquid test sample for a plurality of components. Each of the filaments,which are preferably cotton threads, is used to bind a differentallergen.

Another example of presently available in-vitro devices is given in U.S.Pat. No. 4,567,149 (Sell et al.), which discloses an apparatus includinga well which contains a plurality of elongated strips. Each strip iscoated with a separate assay binding component such as an antigen orallergen. The well is adapted to contain a liquid specimen forincubation with the strips. After the incubation process the liquidspecimen is removed and the binding reaction which occurred on eachstrip is determined by known methods.

Still a further device which may be used for effecting a plurality ofantibody-antigen reactions simultaneously in one operation is disclosedin European Patent Application No. 0 063 810 A1 (Gordon et al.). TheGordon reference teaches a device for carrying out immuno-assays whichcomprises a solid porous support, preferably made of a nitrocellulosematerial, having antigens and/or immunoglobulins bound thereon by directapplication, thereby forming an array of test areas. The array thusformed comprises a plurality of dots or lines of the antigen and/orimmunoglobins.

Various systems are available which may be used in conjunction with theabove-described multiple component binding assay systems to quantify thereactions which occur on the carriers. For example, U.S. Pat. No.4,558,013 (Marinkovich et al.) discloses an apparatus (which may be usedin conjunction with a device such as the one taught by Sell et al.) inwhich a carrier with an uncoated reference region is used to manuallyproduce a strip of photographic film having a linear array of spots orstripes. Each spot or stripe on the film has an optical densityindicating the magnitude of the binding reaction on a particular teststrip or thread. A scanning densitometer is then used to successivelymeasure the optical density of each film strip, thereby providing aquantitative measure of a patient's reaction to the various allergens.

Another device which may be used with the above-described multiplecomponent binding assay systems to quantify the reaction of eachspecific allergen is taught in U.S. Pat. No. 4,510,393 (Sell et al.)which discloses a portable photo chamber which is used to manuallyphotographically record the magnitude of a chemical reaction evidencedby the emission of radio-activity by a substrate labelled with aradioactive tracer.

Although these methods provide advantages over previously availablein-vitro methods, and over the in-vivo methods, they are not withoutlimitations. One major limitation is the fact that the methods foreffecting and measuring the reactions on the above-described multipletest spot devices require an extensive amount of manual manipulation bythe physician or technician performing the test, which increases thetime, cost, and risk of error associated with such tests. For example,known in-vitro procedures require that the multicomponent biologicaltest carriers be manually contacted with the liquid sample beinganalyzed, removed from the liquid sample, washed, and then incubatedwith a solution typically containing a labeled second antibody that isreactive with human IgE class antibodies. Subsequently the carrier mustbe manually removed from the solution and the magnitude of the resultingbinding reaction on the solid phase be then determined byautoradiographical analysis in conjunction with densitometric analysisas proposed by Marinkovich, by fluorometry, or by other knowntechniques.

In addition, the washing step identified above normally comprises amulti-step procedure including removing waste fluid (for example usedreagent or sample solution), adding wash solution, agitating the washsolution for a predetermined time period, removing used wash solution,adding more wash solution, and repeating the cycle two or more timesbefore adding the next reagent. If a number of patient samples are to beanalyzed simultaneously, the hands-on time requirements are furthermagnified. For instance, if ten patient samples were to be analyzed,each washing step alone could involve performing 90 washes. This wouldprobably require a minimum of approximately 30 minutes hands-on time forthe technician or physician for each washing step required.

A number of analyzers for automatically analyzing a plurality ofbiological samples are known. Such analyzers typically include automatedapparatus for providing wash, reagent, and sample fluids, and automatedapparatus for measuring the results of the tests on the samples. See,for example, U.S. Pat. Nos. 4,427,294 (Nardo); 4,451,433 (Yamashita, etal.); 4,406,547 (Aihara); 4,634,575 (Kawakami, et al.); 3,964,867(Berry); and 4,061,469 (DuBose).

Although these analyzers generally automate the analysis of a pluralityof biological samples for the presence of a particular substance, noneare suitable for carrying out the procedures required to simultaneouslyanalyze a plurality of patient samples in a plurality of test cartridgeseach containing a plurality of different test sites and each adapted tosimultaneously perform a complete panel of tests on a single sample.

Available systems have still further limitations. For instance, theaccuracy of test results derived from devices such as those disclosed byGordon et al. may be less than optimal. Since the test dots in theGordon et al. device are formed by direct contact of the specificallergen with the nitrocellulose without an effective means forconfining or isolating the allergen to a specific area, the accuracy andreliability of the results achieved with this device are affected.Specifically, if the dots are arranged in close proximity to each otherthere is a possibility that an allergen from one test dot will migrateonto a neighboring test dot when the allergen is applied to the support.This migration adversely affects the accuracy of the determination ofthe patient's reaction to the allergen associated with the neighboringtest dot. Second, since the specific allergens are not confined to apredetermined area, the concentration of allergen will vary from dot todot on each carrier and from carrier to carrier. As a result, dependingon the detection technique employed, dot to dot variations in opticaldensity or in the intensity of optical or other radiation resulting fromthe binding reactions on a dot will occur in dependence on the area overwhich the allergen initially dispersed during the initial contact withthe support. Such variations have a substantial adverse affect on theuniformity and repeatability of test results.

Therefore, in view of the above, it is a general object of the presentinvention to provide a biological sample analyzer which may be used toautomatically and simultaneously carry out a panel of tests on each of aplurality of patient samples.

It is a more specific object of the present invention to providereaction cartridge means which are adapted for use in such an analyzerto simultaneously test a patient sample for a plurality of differentcomponents with a single addition of patient sample and selectedreagents and which provides test results accessible by an optical readerdirectly on the reaction cartridge.

It is also a more specific object of the present invention to providereaction cartridge conveying means for such an analyzer including meansto accurately and uniformly position a plurality of such cartridges inthree separate dimensions so that an optical reader can accurately anduniformly read the results of a plurality of tests on each of aplurality of patient samples.

It is also a more specific object of the present invention to providemeans adapted use with such an analyzer to provide access to a largevolume of predetermined assay calibration data, such means preferablyincluding reaction cartridge means provided with code means to accesscorresponding assay calibration data in a data storage means.

SUMMARY OF THE INVENTION

To achieve the foregoing and other objects and in accordance with thepurposes of the present invention, automated apparatus for testing eachof a plurality of biological samples for a plurality of selected assaybinding components simultaneously is provided.

The apparatus includes reaction cartridge apparatus having a pluralityof test sites each bound with a preselected first assay bindingcomponent which is adapted to capture a specific second assay bindingcomponent of interest in a biological sample.

Cartridge conveying apparatus or rack means having a plurality ofmounting locations each adapted to hold a reaction cartridge isoperative to selectively convey the reaction cartridges to positions atwhich selected biological samples and reagent fluids are introduced tothe test sites on each cartridge to simultaneously carry out apreselected panel of tests on each sample. The cartridge conveyingapparatus is further operative to selectively convey the reactioncartridges to a test result reading position.

Test result reader apparatus is provided to read the results of thetests directly from the test sites on the cartridges at the readingposition.

In one aspect of the invention, a reaction cartridge is provided whichincludes a plurality of isolated biological sample test sites containedwithin a reaction well which is adapted to contain a biological sampleto be tested. The reaction well is configured to provide direct opticalaccess to each of the test sites. The cartridge is further provided withlock means which cooperate with lock means on a cartridge-conveyingcarousel rack to position and lock the cartridge in three dimensions ina predetermined position on the rack. The rack preferably includes aplurality of openings each adapted to receive a cartridge.

In another aspect of the invention, apparatus is provided for providingassay calibration data adapted for use in assaying biological samples.Predetermined assay calibration data for normalizing the results of atleast one assay with respect to at least one predetermined standardvalue includes a first code for identifying the at least one assay towhich the calibration data corresponds. The calibration data is enteredinto a location in a data storage apparatus. Apparatus such as areaction cartridge, which is adapted for use in carrying out at leastone assay, includes a second code corresponding to the at least oneassay. Apparatus responsive to the second code is provided forcorrelating the second code to the first code to access the calibrationdata in the storage apparatus.

The foregoing objects, advantages and novel features of the invention aswell as others will become apparent to those skilled in the art uponexamination of the following detailed description of a presentlypreferred embodiment of the invention in conjunction with the appendeddrawings. The objects and advantages of the invention may be obtained bymeans of the instrumentalities and combinations particularly pointed outin the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a preferred embodiment of the biologicalsample analyzer of the present invention.

FIG. 2 is a perspective view of a preferred embodiment of a reactioncartridge and a partial cutaway view of a preferred cartridge-conveyingcarousel of the present invention.

FIG. 3 is a top plan view of a preferred embodiment of the carousel ofFIG. 2 illustrating the preferred cartridge positioning means of thepresent invention.

FIG. 4 is a bottom plan view of the carousel of FIG. 3.

FIG. 5 is an enlarged top plan view of a preferred embodiment of thereaction cartridge illustrated in FIG. 2.

FIG. 6 is a partial sectional view through lines 6--6 showing thecartridge mounted in the carousel illustrated in FIG. 3.

FIG. 7 is a partial sectional view through lines 7--7 showing thecartridge mounted in the carousel illustrated in FIG. 3.

FIG. 8 is a magnified view, partially cutaway, through lines 8--8showing sample test sites in a preferred laminate structure of the testcard of the present invention.

FIG. 9 is a cutaway side elevational view of a preferred embodiment ofthe boom arm and drive arrangements of the present invention.

FIG. 10 is a top plan view, partially in phantom, illustrating the rangeof motion of the preferred boom arm of the present invention.

FIG. 11 is an exploded perspective view, partially cutaway, of apreferred embodiment of a spring plate mounting arrangement for the boomarm and carousel drive motors of the present invention.

FIG. 12 is a sectional view of the optical reader head of the presentinvention illustrating a preferred embodiment of an optical reader forreading test results.

FIG. 13 is an electrical schematic diagram illustrating a preferredembodiment of a signal processing and control circuit for use with theoptical reader of FIG. 12.

FIG. 14 is a block diagram illustrating a preferred embodiment ofapparatus of the invention for providing assay calibration data for usein testing patient samples.

FIG. 15 is a block diagram illustrating a preferred system controlarchitecture of the present invention.

FIG. 16 is an exploded view of a preferred embodiment of the well covercomprising a part of the reaction cartridge of the present invention.

FIG. 17 is an alternate preferred embodiment of the well covercomprising a part of the reaction cartridge of the present invention.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Referring now to the drawings and specifically to FIGS. 1-10, abiological sample analyzer 10 includes a processing chamber 11 in whichto test biological samples. A chamber door 12 is preferably hingedlymounted to the analyzer 10 overlying the chamber 11 to selectively closeoff and provide access thereto. Inset into the chamber door 12 is atranslucent viewing window 14 which allows an operator to view theactivity within the processing chamber. The window 14 preferablyincludes a reagent addition port 16 through which reagents can beintroduced into the chamber 11 without opening the chamber door 12.

The processing chamber 11 contains a holding rack, preferably in theform of a rotatable carousel 18 which serves two primary purposes.First, the carousel 18 comprises means for holding and conveyingreaction cartridges 80 in order to position the cartridges to receivesample and selected reagents, to provide agitation required forprocessing the samples and reagents, and to position the cartridges forreading test results therefrom. Second, the carousel 18 functions as avery precise optical bench, accurately positioning each reactioncartridge 80 relative to an optical reader 32, which is described indetail below, to facilitate accurate and repeatable reading of testresults directly from the cartridges 80. Positioning and alignment ofthe cartridges 80 is preferably accomplished using a three-point systemassociated with each cartridge 80. The three-point alignment system ismore fully described below. The carousel 18 also preferably includesoptical positioning means which is used to provide precise alignment ofthe carousel and the optical reader 32 in a manner described in detailbelow.

As illustrated in FIG. 1, the carousel 18 is preferably disposed on atilted axis. Rotation of the carousel 18 about this tilted axis providesdesirable agitation of the fluids in a reaction well 86 of each reactioncartridge 80 thereby promoting faster and more complete reactions andallowing the use of smaller volumes of sample and reagents than haspreviously been possible. Rotation is preferably accomplished at a speedless than 25 rpm to avoid the effects of centrifugal force. Applicantshave successfully used rotational speeds in a range between 7 to about20 rpm. The carousel 18 is preferably tilted such that when a reactioncartridge 80 is at the rear of the analyzer 10, the reaction cartridge80 is at the top of the tilt, thereby forcing fluid to collect at oneend of the reaction well 86 (towards the top of the reaction cartridgeas shown in FIG. 5). As the carousel 18 rotates, fluid in the reactionwell 86 flows across the surface of a test card or panel 82 toward theother end of the reaction well until the reaction cartridge 80 reachesthe bottom of the tilt (adjacent to the front of the analyzer). Thefluid motion is then repeated in the opposite direction as the carousel18 continues to rotate the reaction cartridge 80 back toward the top ofthe tilt, thereby providing desirable fluid agitation.

The tilt of the carousel 18 may be implemented by any conventionalsupport structure, but preferably is implemented by mounting thecarousel 18 on a base, which is inclined at the desired angle byconventional support means. A tilt angle of approximately 10° ispresently preferred but, as will be recognized by those skilled in theart, the tilt may be within any appropriate range, for example 5°-20°,which provides the desired agitation.

As described above, in manual testing procedures, washing of reactionsupports or containers is by far the most tedious chore for the personperforming the test. The presently preferred biological sample analyzer10 of the present invention eliminates these tedious operator steps byautomating the washing function using the combination of amicroprocessor-controlled wash/waste unit 20 and the tilted carousel 18.

The wash/waste unit preferably comprises a dual chamber container whichincludes a chamber 22 for holding wash solution, and a chamber 24 whichprovides containment for waste fluid aspirated from the reactioncartridges 80. A wash manifold 26 functions both as a cover for thechambers and as a mount for wash tubing 23 and waste tubing 21. Thecover 26 is preferably provided with suitable sealing means to preventevaporation of the fluids contained in the wash and waste chambers 22and 24.

In a preferred embodiment, a liquid level sensor (not shown) may beassociated with the waste chamber 24 to detect when the waste chamber 24is full and provide a detection signal. An optical sensor (not shown)may also be provided to generate a detection signal when the cover 26 isnot in an appropriate position (such as when the cover and/or thecontainer wash/waste container are removed).

Wash and waste fluids are caused to flow through wash and waste tubing23 and 21 by means of peristaltic pumps 25 and 27. The first peristalticpump 25 is operative to deliver wash solution from the chamber 22through tubing 23 while the second pump 27 is operative to aspiratewaste fluids through the tubing 21 and into the waste chamber 24. Forreasons which will become apparent, the waste pump 27 is preferablyoperated to generate a higher flow rate than the wash pump 25. Thus, thepin 29 of the wash pump 25 is disposed at a radius less than the radiusof the pin of the waste pump 27. The selection, construction, andoperation of suitable peristaltic pumps is well known to persons skilledin the art and a detailed description is not necessary to a completeunderstanding of the invention.

Wash and waste fluids are preferably introduced into and removed fromthe reaction wells 86 of reaction cartridges 80 mounted on carousel 18by means of a fluid probe 28 which is connected with the wash and wastetubing 23 and 21 and which is mounted proximate the free end of ahorizontal, pivotally-mounted probe arm 33. In a preferred embodiment,fluid access to a reaction cartridge 80 is provided when the carousel 18conveys the reaction cartridge 80 to a preselected wash position(preferably around the one o'clock position on the carousel 18 as viewedfrom the front of the biological analyzer 10 in FIG. 1). At thisposition, the tilt of the carousel 18 causes any fluid in the reactionwell 86 to gravitate towards a corner of the reaction well 86. The probearm 33 is rotated so that the fluid probe 28 is positioned above thereaction well 86 of the reaction cartridge 80. The probe arm 33 thenpivots downwardly, causing the probe 28 to dip down into the corner ofthe reaction well. Pump 27 or 25 is then operated to either aspiratefluid from or introduce fluid into the reaction well 86.

Details of the assay protocol are discussed hereinafter. Upon completionof a panel of tests, the test results are preferably read by an opticalreader system which is more fully described below, but which generallyincludes an optical reader 32 and an associated control and signalprocessing circuit. Briefly, the optical reader 32 includes a source ofoptical radiation, an optical detector, and an array of lenses,apertures and filters. The optical reader 32 is preferably mounted in areader head which is in turn disposed proximate the free end of ahorizontal rotatably-mounted optical reader arm 35. In order to read atest result from a test site 84 on a reaction cartridge, the reactioncartridge 80 is conveyed by the carousel 18 to a predetermined readingposition. The reader arm 35 is then rotated out over the reaction well86 of the reaction cartridge 80 until the test site 84 to be read isaligned directly under the optical reader 32. The optical source of theoptical reader 32 then emits a beam of optical radiation onto a smallportion of the test site 84 and the optical detector converts theintensity of the optical radiation reflected by the diffuse surface ofthe test site into an electrical signal. The signal is then processed toobtain the optical density value of the test site 84 which is directlyrelated to the concentration of a binding component of interest in thebiological sample which is specifically reactive with the capturereagent or binding component disposed on the test site. Undermicroprocessor control, the carousel 18 and optical reader arm 35 movein cooperation to sequentially position the optical reader 32 over eachselected test site 84 until all selected test sites 84 have been read.

As shown in FIG. 1, the biological sample analyzer 10 preferably alsoincludes a keyboard 34, which may be used by an operator to enter dataand instrument function commands. The analyzer also preferably includesa conventional display 36 and printer 38 which may be used to prompt theoperator to take action during a test cycle and to provide a record oftest results.

The keyboard 34 preferably includes numeric keys that allow the user toenter data such as assay calibration data or the patient I.D. numberassociated with a certain reaction cartridge 80. The keyboard 34 alsopreferably includes instrument function keys including for example anENTER key which is operative to enter data from the keyboard, a RUN keywhich is operative to initiate or resume a test procedure and an INDEXkey which is operative to rotatably advance the carousel 18 by oneposition. Other keys which allow keyboard data to be cleared or whichprovide control commands for the printer (such as to eject paper or topause the test operation) may also be provided.

As mentioned above, a controlled test environment is preferably providedin the processing chamber 11. During an exemplary enzyme immuno assaywhich is described in detail below for example, the temperature in thechamber is preferably maintained at approximately 35° C. Temperature inthe processing chamber 11 is suitably maintained at a selected level bymeans of one or more conventional electric coil heaters, fans,temperature sensors, and a temperature control curcuit in a manner wellknown to persons skilled in the art. Most simply, for example, thecontrol circuit would operate to compare the analog voltage across thethermistor with an analog reference voltage corresponding to the desiredtemperature. If the thermistor voltage were below the reference voltage,the control signal would issue a signal to turn on the heater. The fanwould operate to continuously circulate air in the processing chamber11. In a more preferred embodiment, however, a microprocessor may readthe temperature from the temperature sensors at predetermined intervalsand control the heater to maintain the temperature at the desired level.

The locations of the various temperature control components are notcritical. However, it is preferable that any fans be mounted or the airfrom the fans be directed in such a way as to avoid directly circulatingacross the optics reader 32 and optical reference means 70 which aredescribed in detail below in order to minimize depositing debris whichmay affect the optical measurements of test results. The selection,construction and operation of the various temperature control componentsare well known to persons skilled in the art and further detailedexplanation thereof is not necessary for a complete understanding of theinvention.

In addition to the temperature control components described above aconventional electrical resistance type heating strip 31 may be providedin the reaction chamber 11 proximate to and overlying the rotationalpath of the carousel 18 to apply additional heat to reaction cartridges80 as they are rotated on the carousel 18. Use of such a heating strip31 is particularly advantageous in preventing condensation from formingon the well covers 90 of the reaction cartridges 86 which condensate canaffect the concentration of fluids in the reaction wells 86, adverselyaffect test results, and constitutes a biohazard.

In a particularly preferred embodiment, the biological sample analyzer10 also includes optical code reader means 306 which may be aconventional optical bar code reader wand and associated processingcircuitry 308 (illustrated in FIG. 14). As described in detail below,the optical code reader means 306 is used to particular advantage toenter large amounts of assay calibration data into the biological sampleanalyzer 10, which data is then used in the preferred embodiment tonormalize the test results obtained from various test sites 84 onvarious reaction cartridges 80. If desired, a storage compartment 40 canbe provided to store the optical code reader means 306 when not in use.

CAROUSEL AND REACTION CARTRIDGES

Referring now to FIGS. 1-7, a more detailed description is given of thereaction cartridges 80 and the carousel 18. As best illustrated in FIG.5, the preferred reaction cartridge 80 includes a test card 82 whichincludes an array of test sites 84 preferably in close proximity to eachother. The test card 82 is contained within a reaction well 86 which isdefined by a well wall 88. The reaction well 86 is preferably providedwith a removable, preferably transparent well cover 90 which preferablyincludes a reagent port 92 to facilitate the delivery and removal offluids from the reaction well 86.

The well cover 90 is preferably made of one or more layers of a thintransparent material with fairly resilient properties, such as apolyester film. A suitable polyester film is commercially available asMYLAR. The port 92 preferably is defined by multiple slits in the wellcover 90. The slits are preferably disposed in one of the lower cornersof the reaction well 86 and arranged to define a generally Y-shapedport. Since the cover 90 is made of a fairly resilient material thisarrangement provides a self-sealing port. The cover 90 is preferablyremovably adhered to the top of the well wall 88 using a suitableadhesive.

As described in more detail below, to further enhance the sealingcapabilities of the port 92 of the cover 90, the port 92 preferablyincludes a second flap system formed in a second layer of polyester filmand attached to the underside of the first layer of polyester film ofthe cover 90.

A first preferred embodiment of the dual layered cover including thesecond flap system as illustrated in FIG. 16. The cover 90 includes afirst layer 400 bonded to a second layer 402 by a suitable adhesive. Thefirst layer 400 includes three slits which intersect at a single pointand are configured in a generally Y-shaped arrangement. The Y-shapedslit arrangement defines a first layer port 406 with a first hinged flaparrangement. The second layer 402 includes a pair of slits 412, 414configured in a generally V-shaped arranged which define a second layerport 408 with a second layer hinged flap arrangement. As illustrated inFIG. 16, the first layer flap arrangement and the second layer flaparrangement are disposed such that the slits of each port 406, 408 donot directly line up. In this manner the flap of the second layer 402seals the slits of the port 406 in the first layer 400. The flap areasof both layers contain minimal or no effective adhesive to insure freeoperation.

FIG. 17 illustrates another preferred embodiment of a dual layered cover90 including a second flap system. The first or top layer 401 has aY-shaped port 43 configuration similar to the port 406 of the firstlayer 400 of the embodiment illustrated in FIG. 16 and discussed above.The second layer 404 includes slits 416, 418 and 420. The slits 416 and418 are disposed such that they define a generally Y-shaped slitarrangement. The slits 418 and 420 are disposed such that the two slits418 and 420 define a generally V-shaped arrangement. The three slits416, 418 and 420 define a second layer port 410 with a second layer flaparrangement. As with the embodiment illustrated in FIG. 16, the flaparrangement of port 403 and the flap arrangement of the port 410 aredisposed such that the slits of each port 403, 410 do not directly lineup.

With the preferred embodiments of the port 92 illustrated in FIGS. 16and 17, when the probe or syringe needle, for example, enters thereaction well 86 through the slits at the Y-shaped port in the first ortop layer, the hinged flap in the bottom or second layer is pushed down,thereby opening the port 92. When the probe 28 or needle is withdrawn,the hinged flap returns to its normal position, sealing the slits of theport 92 and thereby further enhancing the sealing capabilities of theport 92.

The reaction cartridge 80 also preferably includes code means 94 such asan optical bar code which is attached to or printed directly on the flatsurface 91 of the reaction cartridge 80. The bar code 94 is adapted tobe read by the optical reader 32 or by other conventional optical readermeans. In a particularly preferred embodiment, the bar code 94 includesa lot code which is advantageously used to access stored assaycalibration data corresponding to the particular reaction cartridge 80being tested. A more detailed description of this feature of theinvention is given below.

The reaction cartridge 80 also preferably includes a panel 96 which mayinclude information such as the expiration data of the particularreaction cartridge, the lot number of the particular panel of thecapture reagents or assay binding components used to manufacture thereaction cartridge, and a section on which the operator may manuallyrecord information such as a patient I.D. number and/or the date of thesample being tested.

Referring now specifically to FIGS. 2-4, the carousel 18 includes aplurality of openings 98 which are adapted to receive the reactioncartridges 80. Lock means are provided on the carousel 18 and thereaction cartridges 80 which cooperate to precisely position and lockeach reaction cartridge 80 in the opening 98 in a precise predeterminedposition. Such positioning is preferred in order to minimize variationsin the positioning of the cartridges relative to the optical reader 32and the attendant position-induced variations in the readings of thetest results from cartridge to cartridge.

Preferably, a three-point system is used to position and lock eachcartridge 80 in an opening 98. The three-point locking system includesmeans for positioning and locking the reaction cartridge 80 in each ofthree predetermined dimensions, i.e., in a radial direction, acircumferential direction, and a vertical direction. A radial directionis defined here as a direction which extends radially from the center ofthe carousel 18 and a circumferential direction is defined here as adirection around the circumference of an imaginary circle which isconcentric with the carousel 18.

Preferably the means for positioning a cartridge 80 in the verticaldirection includes a set of tabs 100, 102 and 104 which are mounted atpredetermined vertical distances above the surface of the carousel 18and which are adapted to engage the top of the flat horizontal surface93 of the reaction cartridge 80. The positioning means preferablyfurther includes means for vertically biasing the reaction cartridge 80such that the surface 93 firmly engages the tabs 100, 102 and 104.

The preferred vertical biasing means includes spring clips 106 which arepreferably integrally formed in the surface of the carousel 18 bymolding or another suitable process of manufacture. The spring clips 106preferably include a first angled face 108 and a second angled face 110.The first angled face 108 is adapted to engage a vertically extendingtransverse rib 112 on the bottom of the reaction cartridge 80 as thereaction cartridge 80 is inserted radially into the opening 98 and tourge the spring clip 106 downwardly to allow entry of the cartridge. Thesecond angled face 110, which is preferably oppositely inclined to thefirst angled face 108, is adapted to engage the rib 112 of the reactioncartridge 80 after it passes over the face 108 to lock the cartridge 80in position. The angled face 110 biases the rib 112 and thus thecartridge 80 upwardly such that the flat horizontal surface 93 firmlyengages the tabs 100, 102 and 104. The angled face 110 also functions tolock the reaction cartridge 80 in the predetermined vertical position.

Preferably, the ribs 112 are made of the same material as the base ofthe reaction cartridge 80 and are formed as an integral componentthereof, for example by a conventional plastic molding process. Inaddition to ribs 112, as best illustrated in FIG. 6, the preferredreaction cartridge 80 also includes a flat substantially-vertical wall114 at the front of the cartridge 80. The vertical wall 114 is adaptedto engage a substantially-vertical mating wall portion 116 on thecarousel 18. The carousel wall portion 116 is preferably arranged as acircumferentially extending wall portion as best seen in FIGS. 2 and 3.The mating walls 114 and 116 preferably include at least one contactpoint tangential to the circumferentially extending wall portion 116.The angled faces 110 of the spring clips 106 function to urge or biasthe cartridge 80 in a forward or inward radial direction such that themating walls 114 and 116 firmly engage at the contact point and thecartridge 80 is locked in a precise predetermined radial position.

The means for positioning a cartridge 80 in a circumferential directionpreferably includes at least one and preferably a plurality ofcircumferential contact points between the carousel 18 and the reactioncartridge 80 and means for circumferentially biasing the cartridge 80 tofirmly engage the carousel 18 at these circumferential contact points.In a preferred embodiment, the carousel 18 includes vertical walls 122which are preferably formed as an integral component of the carousel 18,for example by a conventional plastic molding process. The vertical wall122 includes a radially-extending portion 124 which includes a firstcircumferential contact point 118 and an angled portion 126 whichincludes a second circumferential contact point 120. The preferredreaction cartridge 80 includes a wall which is shaped to mate with thevertical wall 122 and which includes a radial portion 128 adapted toengage the first contact point 118 and an angled portion 130 adapted toengage the second contact point 120.

As illustrated best in FIGS. 2 and 7, a vertically-extending spring clip132, which is preferably formed as an integral component of a secondangled portion 131 of the wall 122 of carousel 18, is adapted to engagean angled side wall of the cartridge 80 opposite the wall 130 and tobias the cartridge 80 in a circumferential direction against the contactpoints 118 and 120. The spring clip 132 preferably includes thepreviously described tab 102 as an integral component thereof.

To facilitate the alignment of the cartridge 80 when it is beinginserted into an opening 98 on the carousel 18, a secondradially-extending wall oppositely disposed to the wall 122 is providedon the carousel 18 and a corresponding mating wall is provided on thereaction cartridge 80.

As best shown in FIG. 6, the cartridge 80 preferably includes a set ofribs 138 which underlie the surface 93 and which provide a grippingsurface for an operator to insert the cartridge into or remove thecartridge from the carousel 18.

As best illustrated in FIG. 3, the carousel 18 preferably includesoptical positioning means 140. The optical positioning means 140preferably comprises a parallelepiped structured 142 mounted atop avertically extending base. Although other shapes could be used forstructure 142, the parallelepiped structure is preferred because theedges of such a structure appear to be normal to the arcuate path ofmotion of the optical reader 32. The base preferably extends verticallyfrom the surface of the carousel 18 a predetermined distance such thatthe top of the parallelepiped structure 142 is disposed at the sameelevation as the surface of a test card 82 when a reaction cartridge 80is in the locked position in the carousel 18.

The optical positioning means 140 is advantageously used to determine azero position reference from which the precise position of each testsite 84 of each reaction cartridge 80 on the carousel 18 may be computedfor precise access by the optical reader 32. In order to preciselydetermine the positions of the test sites 84 (or any other location on areaction cartridge), the optical reader arm 35 rotates the opticalreader 32 to scan the optical positioning means 140. The optical reader32 scans the parallelepiped structure in both radial and circumferentialdirections. On each scan, the optical reader 32 takes a plurality ofuniformly spaced optical reflection intensity readings in a mannerdescribed in detail below. By comparing the intensity of sequentialreflection readings, the precise locations of the edges of theparallelepiped structure are determined. In the preferred embodiment,the locations of the edges are represented as a number of stepper motorcounts of the boom arm 30 and the carousel 18 from predetermined homepositions. After the edge locations of the parallelepiped structure aredetermined, the center of the parallelepiped structure 142 is easilyderived by dividing the distance between opposite edges by two andadding the result to the number of steps between the home position ofthe boom arm 30 or carousel 18 and the edge. Knowing the nominaldimension of the carousel 18 and cartridges 80, the position of eachtest site 84 or other location may then be computed relative to the zeroreference coordinates.

As shown in FIG. 4, encoding ring segments 141, which can be read withan opto switch as is hereinafter described in detail, are placed aroundthe carousel 18. The ring segments 141 preferably vary in length toidentify each station.

Preferably, the carousel 18 and cartridge 80 are injection molded from asynthetic plastic material. An acetal material is preferred for thecarousel 18. A suitable material for the cartridge 80 is commerciallyavailable as ABS plastic.

TEST CARD ASSEMBLY

As described above, the preferred reaction cartridge 80 includes areaction well portion 86 which contains a test card 82 and which isadapted to hold a patient sample and selected reagents in contact withthe test card 82 during a test.

Referring to FIG. 8, the test card 82 is preferably a laminate structurecomprising a binding layer 83 adhered to a non-absorbent substrate 85using an adhesive such as a double-sided adhesive film 87. The porousstructure of nitrocellulose has been found to have excellent absorptionand adsorption qualities for a wide variety of fluid capture reagentswhich may be used in connection with the invention and is thereforepreferred for use. Nylon also possesses similar characteristics and is asuitable binding layer. Preferably, a nitrocellulose binding layer 83has an average thickness of about 0.005 inches (127 um avg; ranging fromapproximately 115 to 180 um) and a pore size of about 0.45 um, althoughsome latitude is permissible in these parameters. Preferably, a bindinglayer 83 is also tested for DNA hybridization capacity to bind proteinsor other materials. A nitrocellulose product that has been found tooperate well in the preferred embodiment is available from Millipore(Bedford, Mass.) and is designated HAHY nitrocellulose.

The non-absorbent substrate 85 is suitably a polyester film such asMYLAR plastic having a thickness of approximately 0.002 inches. Thebinding layer 83 is preferably bound to the non-absorbent substrate 85by a double-sided adhesive film 87 such as the adhesive film designatedV-23 or V-29, both of which are commercially available from Flexcon(Spencer, Mass.). An adhesive backed polyester film is commerciallyavailable from several sources, including Flexcon. The entire laminatestructure comprising the test card 82 is about 0.010-0.014 inches thick.

Rolls of the laminate material (nitro-cellulose-adhesive-non-porousbacking substrate) preferred for use in the test card 82 of the presentinvention are made by Millipore (Bedford, Mass.) by combining theirnitrocellulose to the adhesive backed film.

In a preferred mode of manufacture of the test cards 82, the rolls oflaminate material are cut into sheets (not shown) approximately 5 incheswide by 6 inches long. Each sheet is punched with alignment holes forregistration throughout the manufacturing process.

The ultrasound instrument, typically a Branson 4AE or equivalent,includes an ultrasound horn which is configured with a number of raisedcircular ridges. These ridges, which are preferably raised about 0.025inches, apply ultrasonic energy when brought into contact with thelaminate sheet to create circular or annular depressions 89 (bestillustrated in FIG. 8) which go substantially or completely through thebinding layer 83 and may enter into the non-absorbent substrate 85 oradhesive film 87. With a transparent substrate, the depressions 89 aresubstantially optically transparent.

The circular depressions 89 in the binding layer 83 create a pluralityof isolated test sites 84 each composed of binding layer materialencircled by a moat 99 of air space. Each test site 84 is adapted tosupport a reaction between a capture reagent and a specific bindingcomponent in a test sample and to confine the flow of the capturereagent applied to the test site 84 to a specific isolated area. Asshown in FIG. 5, in a preferred embodiment a plurality of test sites 84isolated by surrounding moats 99 are arranged in a predeterminedtwo-dimensional array on the test card 82. Each test site 84 ispreferably approximately 0.1 inches in diameter and each moat 99 isapproximately 0.01 inches across. It is preferable, but not essential,to employ an array wherein the moats 99 considerably overlap oneanother. In this way, the number of test sites 84 on a test card 82 ismaximized. In addition, sensitivity may be improved by reducing theamount of unused binding layer material that competes with the testsites for assay binding components.

It is also preferable to have the depression extend substantiallythrough the porous binding layer 83 so that there are few, if any, poresinterconnecting adjacent test sites 84 through which the capturereagents might flow. Thus, the depression is substantially through thebinding layer and preferably into the adhesive or substrate layer. Thedepth and character of the depression are controlled by selection of theparameters for the ultrasound instrument. For nitrocellulose, theultrasound horn total pressure preferably ranges from about 20 psi to 42psi. The hold time may vary from about 10 ms to about 100 ms; the weldtime may range from about 150 ms to about 400 ms; and the horn frequencyis preferably approximately 40 kHz.

The two-dimensional array of test sites 84 may be achieved in thepresently preferred embodiment by repeated application of an ultrasoundhorn having an array of six annular ridges. A high precision X-Ypositioning table driven by high resolution, computer-controlled steppermotors is used to align the sheet of laminate material under the horn.Advantageously, a single program may be used to control the tablemovement as well as the horn movement. A plurality of holes are formedin the table and are connected to a vacuum source so that the sheet canbe firmly held to the table precisely aligned by placing the alignmentholes over locating pins on the table. Following each application of theultrasound horn, the sheet is moved an incremental amount in the X-axisdirection and the Y-axis direction as is appropriate to generate thepreferred array shown in FIG. 5. Alternative arrays are completelywithin the skill of the ordinary artisan in this area.

When a desired number of arrays are welded onto a sheet of the laminatematerial, the sheet is ready for the addition of one or more selectedcapture reagents or assay binding components. The terms capture reagentand assay binding component are used interchangeably herein and mean anycompound capable of directly or indirectly binding a desired componentfrom a biological test sample. For example, a capture reagent or assaybinding component may include antibodies, antigens, biotin, antibiotin,avidin, lectins, or peptide sequence probes, as well as combinations ofthe above. Typically, reagents are delivered in aqueous solutions, withor without stabilizers, which are discussed in more detail below.

In a presently preferred embodiment, the capture reagent is a specificallergen which binds human IgE class antibodies from a patient serumsample. A fairly comprehensive compilation of such allergens is found inEP 106324 filed in the name of AXONICS. Of course, it is also possibleto employ antibodies as the capture reagent to bind antigens from thepatient's sample. Samples can comprise serum, blood, urine, CSF, salivaand the like.

Advantageously, a different capture reagent is delivered to each testsite 84 so that a single sample can be simultaneously tested for thepresence of binding components specific to each of a panel of differentcapture reagents. Some test sites 84 may have analyte delivered theretoto serve as positive control sites. Other test sites 84 may have noreagents delivered thereto, and can serve as negative control orreference sites. Preferably, from about 1.5 to 4 ul of capture reagentsolution is delivered to each test site 84. This volume exceeds thatwhich can be absorbed or absorbed by the pores of the nitrocellulosecomprising each test site 84 and the excess reagent will bead up overthe test site 84 until it dries and evaporates. If a significantlygreater volume of capture reagent is delivered, however, reagent mayfill the moat 99 and cross to adjacent test sites 84, which could resultin erroneous test results.

Capture reagent may be delivered to a test site 84 by any number ofsuitable delivery methods including reagent jetting, metered airpulsing, positive displacement pump, or by capillary tube lowered to thesurface of the test site 84. In a preferred mode of manufacture, capturereagents are delivered to a plurality of test sites 84 simultaneously.

Positive displacement is the presently used method of deliveringreagents to the test sites 84. In this method, a sheet of test cardlaminate material is vacuum-mounted on an X-Y positioning table similarto that described previously. A precise volume of reagent (about 2 ulfor example) is delivered by a positive displacement syringe pump havinga common driving screw or stepper motor which displaces the plungers ofa plurality of syringes fixed to a support. The syringes empty throughtubing to a plurality of delivery capillary tubes.

Up to 60 (but preferably about 6 to 10) such delivery tubes can bearranged in a fixture which can be raised and lowered using suitablehigh precision drive means in a first pass to deposit droplets ofreagent on the nitrocellulose test sites. The sheet is then moved by theX-Y table and a preselected reagent is deposited on the next array. Whenall the two-dimensional arrays in a sheet of test card material arefilled with the first pass of capture reagents, a new syringe pump setof reagents can be set up for second pass delivery to other test sitesin each of the arrays. Offsetting the delivery tubes and the pass routesso that non-adjacent test sites are spotted during each pass and betweentwo passes minimizes the risk of reagents running together prior todrying.

When each test site 84 on a sheet of test card material has been spottedwith capture reagents (or control reagents) the test sites are allowedto dry thoroughly at room temperature. Drying time may range from about3-72 hours, but is most preferably at least 9 hours.

After drying is completed, the binding layer 83 of the test cardmaterial is preferably "blocked" with a protein coating such asinactivated horse serum or fish gelatin. Blocking masks potentialnon-specific binding sites on the binding layer 83 (including control orreference test sites which have no capture reagent) and eliminatesexcess unbound capture reagent to reduce competition and non-specificbinding. Suitable blocking is obtained during an incubation period ofabout 1 hour at approximately 37° C. and is preferably accomplished intanks with agitation during incubation.

Following blocking, the test card material is washed three times in 10mM Tris buffered saline (TBS) and allowed to dry overnight.

Individual test cards 82 are preferably cut from the dried sheets oflaminate material in generally rectangular shapes adapted to fit thereaction wells 86 of the cartridges 80. Registration holes are also usedto align the sheets with respect to a punch which operates to cut outthe individual test cards 82. In order to optimize assay sensitivity, itis preferred that the cut test cards 82 have minimal unused area ofbinding layer material.

The individual test cards 82 are preferably adhered to the bottomsurfaces of the reaction wells 86 using a two-sided adhesive tape.Precise positioning of the test cards 82 in the wells 86 is criticalsince accurate optical reading of the test sites 84 depends on precisepositioning of the arrays with respect to the zero position referencecoordinates described above. For this reason a vacuum jig apparatus (notshown) is preferably used to insert test cards 82 into the wells 86.Each test card 82 is placed in the corner of a jig abutting twosidewalls. Vacuum drawn through holes in the jig holds the test card 82in place. When the test card 82 is ready for transfer, a movable headaligned with pins on the jig descends over and contacts the test card82. The vacuum is transferred from the jig to the head and the head,with the test card 82 now attached, is moved to a second jig havingidentical alignment pins. The head is lowered over the pins and into thereaction well 86 of a cartridge 80 fixed in the second jig so that thehead precisely registers the test card 82 in the well 86. The headvacuum is then released and the double-sided adhesive tape on the bottomsurface of the well 86 adheres the test card 82 in precise position.

Stabilizers may be used, if desired, to enhance the stability of thecapture reagents delivered to the test sites 84. For example, manyallergens can be stabilized by cross-linking with known agents. Anexemplary listing of such agents and their final preferredconcentrations are given in Table 1.

                  TABLE 1                                                         ______________________________________                                        Crosslinking Agents                                                           Agent                Concentration                                            ______________________________________                                        1-ethyl-3-dimethylaminopropyl                                                                      5 mg/ml/0.4 mg/ml                                        Formalin             4%                                                       Tetrahydrofuran (THF)                                                                              20%                                                      Formalin/THF         4%/20%                                                   Acetic acid/NaOH neutralization                                                                    8% acetic acid                                           Glutaraldehyde       3%                                                       ______________________________________                                    

In addition, some proteins may be stabilized via photocross-linking. Forexample, the use of N-Hydroxysuccinimidyl-4-azidobenzoate (HSAB) and UVlight has been described for linking insulin to nitrocellulose. SeeKakita et al., Diabetes v. 31, pp. 648-652, July 1982. Through the useof these known techniques, capture reagent can be fixed to a test site84 without covalent bonding.

Alternatively, covalent bonding may be used to attach capture reagent toa test site 84, with or without spacer or linker molecules.Functionalization of a binding layer material such as nitrocellulose ornylon can be achieved through a number of mechanisms known in the art.

HORIZONTAL BOOM ARM AND DRIVES

Referring now to FIGS. 9 and 10, a more detailed description of thepreferred boom arm 30 is provided. The horizontal boom arm 30 providesmeans for positioning the optical reader 32 and the wash/waste probe 28over the cartridges 80 in carousel 18. In a preferred embodiment, theboom arm 30 includes both the probe arm 33 and the optical reader arm35, mechanically interconnected. Preferably, a high resolution steppermotor 50 is used to rotate the boom arm 30 to position the opticalreader 32 and the probe 28 in precise increments along an arcuate path.The stepper motor 50 includes a shaft with a fixedly attached piniongear 51 which preferably engages a sector gear 52. The sector gear 52 isin turn fixedly mounted to a shaft 53 which supports a fixed end of theoptical reader arm 35 of the boom arm 30. The shaft 53 is preferablyrotatably mounted to a base plate 54 by conventional bearing means 55.The base plate 54 may be constructed of any suitable material, as forexample, cast aluminum.

Since precise alignment between the boom arm 30 and pinion gear isdesirable, the boom arm 30 and pinion gear 51 are preferably mounted totheir respective shafts by means of a keyed arrangement.

The gear ratio of the sector gear 52 to the pinion gear 51 may be anysuitable ration which provides the precise boom arm positioningrequired, but a ratio of about 13.88/1 with the pinion gear having apitch diameter of approximately 0.375 inches and a total of 18 teeth ispresently preferred.

The carousel 18 is preferably driven by a similar stepper motorarrangement. A carousel stepper motor 56 mounted to the base plate 54,rotatably drives a shaft with a fixedly attached pinion gear 57. Thepinion gear 57 in turn engages a gear 58 which is fixedly connected tothe mounting shaft of the carousel 18, which is suitably mounted to thebase plate 54 via a conventional bearing arrangement. In a preferredembodiment, the gear 58 has a pitch diameter of approximately 0.375inches and a gear ratio of approximately 10/1 with respect to the piniongear 57, which also has a pitch diameter of about 0.375 inches and atotal of 18 teeth. As with the boom arm, the shaft of the stepper motor56 is preferably provided with a keyed arrangement to precisely positionthe pinion gear 57.

The sector gear 52 and gear 58 may be made of any suitable material,such as aluminum or a plastic material such as an acetal. A materialwhich is commercially available under the trade name DELRIN is presentlypreferred for use. Similarly, the pinion gears 51 and 57 may be made ofany suitable material. Presently it is preferred to machine these gearsfrom a stainless steel. Suitable precision stepper motors may bepurchased from several commercial sources. A particularly preferredstepper motor is commercially available from Vexta as Model No.PXC43-03AA.

Since precise positioning of the optical reader 32 with respect to thecarousel 18 is critical, the optical reader 32 must be maintained aslevel with the carousel 18 as possible. Therefore, the shafts whichrotate the boom arm 30 and carousel 18 are preferably carefully mountedwith a predetermined center to center distance therebetween with veryclose tolerance. Additionally, both shafts are preferably carefullymounted to maintain precise vertical alignment between their centeraxes.

If desired, further leveling may be obtained by fixedly mounting a flatpositioning cover plate (not shown) on the carousel drive shaft betweenthe gear 58 and the carousel 18. Such a cover plate preferably includeslocating pins which engage corresponding apertures in the gear 58 toprecisely align the cover plate to the gear 58. The carousel 18preferably includes means which engage key means on the cover plate toprecisely position the carousel on the cover plate. Preferably, meansare also provided to secure the carousel 18 to the cover plate. Thecover plate may be made of any suitable material such as aluminum coatedwith a suitable finish.

As mentioned previously, exact positioning of the boom arm 30 andcarousel 18 is accomplished by counting the number of steps taken by thestepper motors 50 and 56. In a preferred embodiment, exact positioningof the optical reader 32 over any test site 84 of a cartridge 80 on thecarousel 18 is implemented by a combination of movement of the opticalreader 32 in an arcuate path and rotation of the carousel 18 to bring acartridge 80 into a reading position which intersects the path of theoptical reader 32.

Since the precise number of steps taken by stepper motors 50 and 56 iscritical for positioning the reader head 32 over any particular testsite 84, backlash transmitted from the stepper motors 50 and/or 56 tothe gears 52 and 58 may introduce positioning errors which may introduceerror into the optical readings. In order to reduce such backlash,floating mounting means are preferably provided for the stepper motors50 and 56. In a particularly preferred embodiment, for example, a springplate 61 (shown in FIG. 11 with respect to stepper motor 50) is adaptedto provide the pinion gear 51 with a degree of freedom of movement inthe Y-direction while restricting movement in the X-direction. Thespring plate 51 is fixedly mounted to the base plate 54 near the outersection 62. The stepper motor 50 is fixedly attached to the centerportion 63 of the spring plate 61 by conventional fastening means. Inthis manner, the pinion gear 51 firmly engages the sector gear 52 whenthe stepper motor 50 is rotating in a forward direction. The singledirection of freedom of movement provided by the spring plate 51prevents any backlash from the stepper motor 50 from being transmittedto the sector gear 52.

Referring to FIG. 9, the horizontal probe arm 33 of the boom arm 30 hasa fixed end which is mechanically connected to the optical reader arm 35and a free end which carries the wash/waste probe 28. The probe arm 33is preferably pivotally mounted to ears extending downwardly from theoptical reader arm 35 by a pivot pin 65.

In a preferred embodiment, a linear actuator motor 66 having a shaft 67is coupled to a tab 68 which extends upwardly from the probe arm 33.When the shaft 67 is retracted, the probe arm 33 pivots upwardly aboutthe pivot pin 65. When the shaft 67 is extended, the probe arm 32 pivotsdownwardly about the pivot pin 65. Suitable linear actuators motors arecommercially available from a number of sources including Airpax.

As best illustrated in FIG. 9, the outward side portion of the probe arm33 may be left open to allow access to the wash and waste tubing 23 and21, which may occasionally or routinely need to be replaced. In apreferred embodiment, the probe arm 33 is provided with snap or guidemeans to hold the tubes 21 and 23 in place and allow easy removal andinsertion of the tubing.

The tubes 21 and 23 preferably connect to probe block means 31 which maybe mounted proximate the free end of the probe arm 33 in any suitablefashion. The probe block means 31 provides means for both the wash andwaste pumps 25 and 27 to deliver and remove fluid through the samewash/waste probe 28. The probe block 31 prevents contamination of theprobe 28 by providing separate entry points for wash and waste fluidswith the wash point preferably being above the waste point. Both thewash and waste points connect to a common fluid channel which in turnopens into the probe 28. Priming of the probe block means 31 may beaccomplished by delivering wash solution to the probe block andaspirating fluid from the probe block simultaneously. As mentionedpreviously, the waste pump 27 is preferably operated at a greater ratethan the wash pump 25 so that during priming the wash solution neverescapes the probe tip 28 and is passed directly into the waste system.This priming procedure is preferably performed before each test run andis also advantageously performed after changing wash and waste units 22and 24 or before and after tubing changes.

Preferably, the drive arrangements also include home position indicatormeans associated with the carousel 18 and boom arm 30. Such meansadvantageously provide signals indicating when the carousel 18 and boomarm 30 are in their respective predetermined home or starting positions.These means include optical switches (not shown) which cooperate withoptical blocking flags on the carousel 18 and probe arm 30 to indicatewhen the carousel 18 and probe arm 30 pass through predetermined homepositions. The previously described encoding ring segments 141 can serveas home position indicators. Suitable optical switches are commerciallyavailable from Opto Switch, Inc. of McKinney, Tex. as Model No. 0288.These switches are particularly preferred for use with amicroprocessor-controlled analyzer because of their ability to generatea digital output.

OPTICAL READER

In a presently preferred embodiment, the optical reader 32 operates on aprinciple of diffuse reflectance to read test results from the testsites 84 of the reaction cartridges 80. In the diffuse reflectanceprinciple of operation, optical radiation is emitted onto the opticallydiffuse surface of a test site 84 and the intensity of the opticalradiation diffusely reflected by the surface is detected. The densityvaries as a result of color developments by adding conjugate anddeveloping as described in detail hereinafter. The intensity of thediffusely reflected optical radiation is then processed as described indetail below to obtain an optical density value which indicates themagnitude of the binding reaction between a capture reagent or assaybinding component bound to the test site 84 and a second bindingcomponent of interest in the sample tested which has a specific bindingaffinity for the first component. It will be appreciated by personsskilled in the art that alternative optical reading apparatus such as afluorometer, for example, could also be used if desired depending uponthe particular assay chemistry employed.

Referring to FIG. 12, in the preferred embodiment the optical reader 32comprises a reflectometer 160 which is mounted in a reader head 155. Thereaderhead 155 is preferably integrally formed with or, alternatively,mechanically connected to the free end of the horizontal optical readerboom arm 33 as best seen in FIGS. 1 and 9.

In the presently preferred embodiment, reflectometer 160 includesoptical source means 162 and optical detector means 164. The opticalsource means 162 is preferably a solid state device such as a lightemitting diode (LED). In particular, a model H-3000 high intensity redLED which is sold commercially by Stanley and which emits opticalradiation at a nominal wavelength of approximately 660 nanometers at 35degrees centigrade is preferred. The optical detector means 164 is alsopreferably a solid state device such as a photodiode or phototransistor.In particular, a model VDT 020D hybrid silicon photodetector/amplifierwhich is available commercially from United Detector Technology ispreferred.

A cylindrical illumination bore 166 and a cylindrical reflection bore168 are provided in the optical reader head 155 by means of appropriatemachining. The reflection bore 168 is preferably formed vertically sothat its center axis is substantially perpendicular to the surfaces ofthe test sites 84 to be read when the reader 32 is positioned over areaction cartridge 80. The illumination bore 166 is preferably formedwith its center axis being at an acute angle from the vertical centeraxis of the reflection bore 168. In particular, the illumination bore166 is preferably formed with its center axis being at an angle ofapproximately 30 degrees from the vertical center axis of the reflectionbore 168. This arrangement minimizes the amount of optical radiationspecularly reflected from a test site to the optical detection means 164and avoids occlusion of the optical beam emitted by the optical sourcemeans 162 by the wall 88 of the reaction well 86, which wouldundesirably reduce signal levels.

The illumination bore 166 has three concentric sections 166a, 166b, and166c with the upper section 166a having a slightly larger insidediameter than the middle section 166b and the middle section 166b havinga slightly larger inside diameter than the lower section 166c. Anannular shoulder 170 is formed between the middle and lower sections166b and 166c. Lens means 172, which is preferably a circular biconvexlens, has a slightly smaller outside diameter than the inside diameterof the middle section 166b. Lens means 172 is mounted in the middlesection 166b and is supported therein by the shoulder 170 about itsperimeter. An annular ring 174 having an outside diameter slightlysmaller than the inside diameter of the middle section 166b and an innerdiameter selected to minimize optical interference with the lens means172 is mounted in the middle section atop the lens means 172. An annularcompression spring 176 also having an outside diameter slightly smallerthan the inside diameter of the middle section 166b is mountedlengthwise therein atop the annular ring 174 and is supported therebyabout its periphery.

The annular spring 176 extends upwardly into the top section 166a andengages the bottom of a cylindrical LED holder 178 which has an outsidediameter slightly less than the inside diameter of the top section 166aand which is mounted in the top section 166a. The LED holder 178contains a cylindrical LED mounting chamber 180. A concentriccylindrical countersink 182 is formed around the top of the chamber 180to form an annular shoulder 184 therewith. The shoulder 184 is adaptedto support the flanged perimeter of an LED 162 which is mounted in themounting chamber 180. The LED 162 is also preferably adhered in themounting chamber 180 using a suitable adhesive to prevent movementthereof which could adversely affect the reading of test results. TheLED holder 178 is preferably machined of an anodized aluminum and isprovided with a blackened inside surface to minimize optical scattering.However, other processes and materials known to persons skilled in theart and having suitable optical qualities may also be used.

A cylindrical volume aperture 196 having a relatively small diametercompared to the diameter of the bore 166 is preferably provided in thebottom wall of the LED holder 178 to communicate the optical radiationemitted by the LED 162 into the illumination bore 166. The volumeaperture 196 is preferably concentric with the mounting chamber 180 andthe illumination bore 166 and preferably has a longitudinal dimensionthat is several times greater than the inside diameter thereof, which ina presently preferred embodiment may be approximately 0.025 inches.

The use of the volume aperture 196 is particularly advantageous inminimizing optical aberrations commonly associated with LED's. Forexample, typical LED's are known to provide non-uniform sources ofoptical radiation due to the presence of dark spots and/or inaccuratelocation of the semiconductor junction. The volume aperture 196 operatesto collect and diffuse the optical radiation emitted from the lens ofthe LED 162 to provide a more uniform optical source.

In a presently preferred embodiment, adjustment means are provided foradjusting the optical beam emitted from the illumination bore 166. Amounting tab 186 having an opening 188 is integrally formed with the LEDholder 178. The mounting tab 186 mounts in a recess 190 when the LEDholder 178 is mounted in the reader head 155. A threaded bore 192concentric with the opening 188 is provided to engage a threadedfastener 194 such as a screw which may be inserted through the opening188. The threaded fastener 194 may be manually turned to adjust thelocation of the LED holder 178 and thus the LED 162 in the illuminationbore 166.

The reflection bore 168 has two concentric cylindrical sections 168a and168b with the inside diameter of section 168a being slightly greaterthan the inside diameter of section 168b so that an annular shoulder 200is formed between the two sections. Lens means 202, which is preferablya circular plano-convex lens, has a slightly smaller outside diameterthan the inside diameter of section 168a and is mounted thereinsupported about the periphery of its planar side by the shoulder 200.

An annular compression ring 204 also having an outside diameter slightlysmaller than the inside diameter of section 168a is mounted therein atopthe lens means 202 and, in particular, atop the convex side of the lensmeans 202. The inside diameter of the compression ring 204 is preferablydimensioned to minimize optical interference with the lens means 202.

An elongated annular insert 206 having an outside diameter slightly lessthan the inside diameter of the section 168a and an inside diameterapproximately the same as the ring 204 is mounted lengthwise in section168a atop the annular ring 204. The insert 206 is preferably black andis designed to cover substantially the entire exposed inner surface ofthe reflection bore 168 in order to minimize optical scattering therein.The inside surface of the insert 206 is advantageously provided withthread-like discontinuities which further assist in this function. Inthe preferred embodiment, the insert is molded or machined of a blackplastic, preferably a plastic sold commercially under the trade nameDELRIN, although other materials having suitable optical properties mayalso be used.

Preferably, a circular optical filter 208 having an outside diameterslightly less than the inside diameter of the section 168a is mountedtherein atop the insert 206. The optical filter 208 is preferably a red,Schott glass, band-stop filter which is substantially transmissive onlyto optical radiation having wavelengths above approximately 630nanometers.

A cylindrical detector well 210 having an inside diameter greater thanthe inside diameter of the section 168a and concentric with section 168ais preferably formed around the top of section 168a. An annular apertureelement 212 having an outside diameter slightly less than the insidediameter of the detector well 210 is mounted therein overlying theoptical filter means 208. The optical detector means 164 extends insidethe detector well 210 with its optically active surface facing theaperture element 212. In a preferred embodiment, the optical detectormeans 164 is mounted directly to a printed circuit board (not shown)which overlies the detector well 210.

The circular aperture formed by the inside diameter of the annularaperture element 212 is preferably concentric with the reflection bore168 and the detector well 210. The diameter of the aperture determinesthe size of the area of a test site 84 from which reflected opticalradiation is introduced to the optically-active area of the opticaldetector means 164. Preferably, the aperture is selected to have adiameter slightly larger than that of the optical beam which impinges onthe test site to provide a slightly increased depth of field. Thisfeature advantageously reduces the sensitivity of the optical detectoroutput signal to minor variations in vertical distance between varioustest sites and the optical reader 32. In addition, the diameter of theaperture is preferably selected to allow reflected optical radiation toimpinge on substantially the entire optically-sensitive surface of theoptical detector means 164 in order to maximize the output signal levelof the optical detector means 164.

It is possible that when the optical reader 32 is positioned with thereflection bore 168 over a test site 84 located adjacent to the wellwall 88 of a cartridge 80, the wall 88 can occlude the optical beamemitted from the illumination bore 166 and adversely affect the readingof the test site. This possibility can be prevented by offsetting theillumination bore 166 radially from the reflection bore 168 toward thefree end of the optical reader arm 33 by a few degrees. For example, ina presently preferred embodiment, assuming a vertical wall 88 dimensionof approximately 0.5 inches, a nominal vertical dimension of the bottomof the reader head 155 of approximately 0.48 inches relative to thesurface of a test site 84 adjacent to the wall 88, and a nominal angleof 30 degrees between the illumination and reflection bores of thereader, an offset of approximately 10.5 degrees has been found suitableto avoid occlusion of the beam.

In order to minimize output signal variations with height, the opticalreader 32 is preferably configured such that the detection aperature 212is defocused at the intersection point 201 of the optical axis of thetwo light paths. For reasons explained below, the preferred target planeof the optical reader 32 lies above the intersection point 201, with theillumination field of the optical beam off-centered toward theillumination side of the optical reader 32.

The collection efficiency of the detector optics is increased by raisingthe target plane toward the optical reader 32 from the intersectionpoint 201. Given the vertical dimension and the angle value of the aboveexemplary arrangement, the best focus of the detector aperature 212 liesapproximately 0.240 inches above the intersection point 201. However, asthe target plane is raised toward the optical reader 32, theillumination field of the optical beam emitted from the illuminationbore 166 moves toward the illumination side of the reader 32, moving theilluminated target away from the area of maximum sensitivity of thedetector optics. At some point, as the target plane approaches theoptical reader 32, the illumination field falls outside of thedetectable area and the detector output signal declines. The point ofmaximum signal is the area of minimum sensitivity to target height. Inthe exemplary arrangement given above, the point of maximum signaloccurs approximately 0.030 inches above the intersection point 201.

In using the optical reader 32, the threaded fastener 194 is preferablyused to adjust the distance between the optical source 162 and the lensmeans 172 to provide a beam of optical radiation at the intercept of theaxes of the illumination and reflection bores 166 and 168 having adiameter of approximately 0.03 inches.

The lens means 202 in the reflection bore 168 collects the opticalradiation reflected in a substantially perpendicular direction by theoptically diffuse surface of the test site 84 lying beneath thereflection bore 168 and projects it onto the surface of the opticalfilter means 208, preferably slightly defocused. In a particularlypreferred embodiment the aperture element 212 is provided with an insidediameter of approximately 0.095 inches so that optical radiation from anarea somewhat larger than the area of the test site impinged upon by theoptical beam is transmitted to the optically-sensitive area of theoptical detector means 164.

Referring now to FIG. 13, a detailed description of a preferredembodiment of a signal processing and control circuit for the opticalreader 32 is provided. Preferred components and component values of thecircuit are as illustrated.

It should be noted initially that the preferred circuit may be embodiedon a conventional printed circuit board using conventional printedcircuit fabrication techniques. In a preferred embodiment, the printedcircuit board is shaped to fit within the optical reader arm 35 and maybe mounted by conventional fastening means in a cavity 215 which extendsinto the reader head 155 above the reflectometer 160 (FIG. 12). In thisembodiment, the top of the optical reader arm 35 is preferably providedas a removable cover 217 which is secured by screws or the like toprovide access to the printed circuit board and the reflectometer 160.

The presently preferred optical reader signal processing and controlcircuit 225 includes means for converting an analog output signal of theoptical detector means 164, which is directly related to the intensityof the optical radiation reflected by a test site 84 or other opticaltarget, to a digital signal for further processing. The preferredcircuit also includes means for controlling the drive signal of the LED162 to control the output intensity thereof. In one preferred embodimentdescribed in greater detail below, this feature is useful to compensatefor variations in output intensity due to temperature variations.

More specifically, in the preferred circuit 225 the analog signal outputby the optical detector means 164 is communicated to the signal inputVIN of A-D converter means 220. A-D converter means 220 is preferably ofthe voltage to frequency type but other known types of A-D convertersmay be used if desired. The A-D converter means 220 samples theinstantaneous level of the optical detector analog output signal presentat the VIN signal input at a rate determined by clock signal CLK IN.Preferably, the CLK IN signal, which may be provided by a crystaloscillator or other known clock signal generator means, has a nominalfrequency of approximately 2 MHz.

The A-D converter means 220 generates output signals on the FOUT signaloutput comprising a digital pulse train having frequency linearlyrelated to the sampled level of the analog signal at the VIN input.

The FOUT signal output is connected to the trigger or clock signal inputTRIG of counter means 222, which is suitably a conventional 16-bitcounter. Counter means 22 is controlled by a microprocessor 315 (FIG.15), which is described in greater detail below, by means of a countenable signal COUNT ENAB and a count reset signal COUNT RESET. The COUNTENAB signal is provided to the counter enable input ENAB and the COUNTRESET signal is provided to the counter reset input RST of the countermeans 222.

In the preferred embodiment, the COUNT ENAB signal is used to define anintegration period during which the counter means 222 counts the digitalpulses generated by the A-D converter means 220. The COUNT ENAB signalmay be generated with a predetermined interval by conventional meanssuch as a monostable multivibrator. However, for additional flexibility,the use of a programmable interval timer (PIT) 344 (FIG. 15) ispreferred. Thus, in a preferred embodiment, the PIT 344 is programmed tocount down a selected interval by the microprocessor 315 which then setsthe COUNT ENAB signal to enable the counter means 222. When the PIT 344times out, the microprocessor 315 responds by resetting the COUNT ENABsignal to inhibit further counting by the counter means 222. Byselecting an integration interval greater than the sample period of theA-D converter means 220, the counter means 222 is operative to integratethe optical detector output signal over time and thereby reduce theeffect of spurious high frequency noise components. In a presentlypreferred embodiment, an integration interval of approximately 25milliseconds is preferred.

Each integration interval corresponds to an optical reading. Followingthe completion of an integration interval, the microprocessor 315 readsthe final count value on the counter outputs D0-D15. The count valuerepresents the intensity of the optical radiation reflected from thesurface of a test site 84 or other optical target integrated over theselected time interval. Prior to initiating each subsequent integrationinterval, the microprocessor 315 generates the COUNT RESET signal toreset the counter outputs D0-D15.

As mentioned previously, in a presently preferred embodiment a highintensity LED is used as the optical source means 162. In order toprevent variations in the temperature of the LED from causing variationsin the LED's output intensity which would adversely affect the accuracyof the optical readings, one preferred embodiment of the circuit 225includes temperature sensing means 226 and LED drive control means 224which is responsive thereto. The temperature sensing means 226 issuitably a thermistor or similar device that generates a signal thevalue of which is related to ambient temperature. Preferably thetemperature sensing means 226 is mounted as closely as possible to theLED. Referring to FIG. 12 for example, the temperature sensing means 226may be mounted in a cavity 183 of the optical reader head 155immediately behind the LED 162. Although not illustrated in FIG. 13 toavoid duplication, A-D converter means and counter means identical toA-D converter means 220 and counter means 222 are preferably provided toconvert the analog signal generated by the temperature sensing means 226to a digital count value which may be read by the microprocessor 315.The LED drive control means 224 preferably comprises voltage-controlledvariable impedance means such as transistor means having a collectorconnected to the cathode of the LED 162 and an emitter connected toground. The level of an LED drive control signal LED CONTROL determinesthe base current of the transistor means which in turn determines theimpedance value of the collector-emitter path of the transistor meanswhich is in series with the LED 162. Alternatively, other controllablevariable level impedance devices could be used.

In this embodiment, prior to initiating each integration interval andtaking an optical reading, the microprocessor 315 reads the digitalvalue representing the temperature of the LED. A table of digital inputvalues for a D-A converter 342 (FIG. 15) which correspond to LED analogdrive currents necessary to maintain the output intensity of the LED ata predetermined constant level at various temperatures is predeterminedempirically and stored in a memory such as RAM 334 (FIG. 15). Themicroprocessor 315 uses the measured digital temperature value as anindex into the table, retrieves the appropriate digital D-A input value,and applies it to the D-A converter 342. The D-A converter 342 in turngenerates a corresponding analog LED drive control signal LED CONTROLwhich is applied to the LED drive control means 224. The LED drivecontrol means 224 responds to the LED CONTROL signal to control thedrive current allowed to flow through the LED and thereby maintain thedesired output intensity of the LED over a certain temperature range.

In a second and more preferred embodiment, the microprocessor 315 uses apredetermined temperature compensation factor to compensateoptically-read reflection intensity values for variations of themeasured temperature from a predetermined reference temperature, forexample 35 degrees centigrade. In this more preferred approach, themicroprocessor 315 applies a predetermined digital value whichcorresponds to a desired output intensity of the LED 162 to D-Aconverter 342. Although the DAC provides flexibility to change the LEDdrive current if necessary or desired by simply varying the digitalinput value, it is not necessary to change the value with changes intemperature because the reflection intensity readings are compensateddirectly for the temperature changes.

Before describing in detail how the temperature compensation factor usedin the more preferred embodiment is computed, attention is directed toFIG. 10 wherein optical reference means 70 is illustrated. Opticalreference means 70 provides a white optical reference which is used as acommon standard against which to normalize the reflection intensityreadings taken from the test sites 84 on the various reaction cartridges80. Preferably, the optical reference means 70 comprises a punched steelpost having a flat top. A ceramic mixture having a preselected optical"whiteness" value is preferably applied to the top of the post and isthen baked on. For example, a white ceramic top matching the NationalBureau of Standards no. 1 white reference swatch is presently preferredfor use. However, it should be noted that the ceramic defines anarbitrarily selected white reference value and that other whitestandards may therefore also be used. The post is preferably mounted tothe analyzer 10 in a location that intersects the arcuate path of theoptical reader 32 so that the reflection bore 168 of the reflectometer160 can be positioned directly over the ceramic top. The post itself ispreferably mounted in such a way as to be vertically adjustable so thatthe vertical distance between the optical reader 32 and the surface ofthe ceramic can be adjusted to equal the vertical distance between thereader head 155 and the surface of the test sites 84 on the reactioncartridges 80. For example, the post can be threaded on its lower halfand screwed into a corresponding threaded receiving bore in the analyzer10.

A cover 72 is also preferably mounted to the analyzer 10 and ispreferably positioned and shaped to overlay the optical reference means70. The cover 72 is adapted to prevent dust particles or other debrisfrom accumulating on the ceramic surface of the reference means andchanging the optical reflectance of the ceramic surface. The cover 72 ispreferably pivotally mounted and biased in a normally closed position.Corresponding tabs (not shown) may be provided on the cover 72 and boomarm 30 so that when the optical reader 32 is rotated into position toread the optical reference means 70, the tabs engage and pivot the cover72 to expose the ceramic surface. When the boom arm 30 rotates away fromthe reference means 70, the cover 72 preferably returns to itsnormally-closed position.

Describing now in detail the preferred process of computing thetemperature compensation factor, it is initially noted that it has beenempirically determined that the output intensity of the LED 162 in thepresently preferred embodiment varies substantially linearly withvariations in temperature over an expected maximum temperature range ofapproximately 30°-40° C. Accordingly, a linear equation which relatesthe LED output intensity to temperature can be derived.

A preferred process of deriving the equation involves first positioningthe optical reader 32 over the reference means 70, taking a darkreflection intensity reading with the LED off, and storing the reading.Next the temperature in the processing chamber 11 is cycled through theexpected range of temperature values and a plurality of reflectionintensity readings are taken of the reference means 70 over the entiretemperature range. Each time a reading is taken the temperature ismeasured. Each reflection intensity reading is netted by subtracting thestored dark reflection intensity reading in order to remove componentsdue to the presence of ambient radiation. The corresponding measuredtemperature and net reflection intensity data pairs are then stored.This procedure is preferably repeated at least two more times togenerate a representative body of data.

Next the corresponding data pairs generated during each temperaturecycle are processed using conventional linear regression techniques toobtain the slopes and intercepts of the best fit linear equations whichdefine a predicted relationship between the reflection intensity of theknown white reference means 70 and measured temperature for each cycle.Each derived equation is then solved to obtain a net reflectionintensity value at 35 degrees centigrade and the slope value of eachequation is normalized to 35 degrees by dividing the slope value by thepredicted net intensity value at 35 degrees. The normalized slope valuesare then averaged and the average slope value, which is expressed inunits of counts per degree, is stored as the temperature compensationfactor.

In a third and even more preferred embodiment, the thermistor 226 of thefirst embodiment is replaced by a second optical detector (not shown).In this embodiment, the second optical detector is mounted directlybehind the LED 162 and generates an analog signal having magnitudedirectly related to the intensity of the optical radiation backscattered from the LED 162. Each time an optical reading is taken, thesignal from the first and second optical detectors are simultaneouslyconverted and integrated over the same integration interval. Then themicroprocessor processes the two count values to form the ratio of themeasured reflection intensity to the back scatter intensity andthereafter treats the ratio as the reflection intensity reading. Sinceboth readings are equally affected by LED 162 temperature variations,the ratio remains constant and provides a temperature-compensatedreflection intensity value. No temperature measurements nor additionalcompensation of the reflection intensity readings is necessary in thisembodiment.

The optical reference means 70 also provides a gray scale reflectancereference value, i.e. optical density value, which is advantageouslyused to calibrate or normalize the gray scale reflectance values whichare derived from reflection intensity readings of the various test sites84 on the various reaction cartridges 80 taken by the optical reader 32.The gray scale reflectance reference value is assigned to the opticalreference means 70 by first reading the reflection intensity value ofthe optical reference means in the manner described in detail above.Next the reflection intensity value of a plurality of optical standardshaving known gray scale reflectance values is read. For example, aconventional optical filter test card having a plurality of opticalfilters, each with a different known gray scale reflectance value may beread. A suitable test card having eight optical filters each with adifferent known gray scale reflectance value is available commerciallyfrom Munsell.

Each of the reflection intensity values read from the optical referencemeans 70 and the optical standards is netted by subtracting thepreviously stored dark reflection intensity of the optical referencemeans 70. Then each of the net reflection intensity values iscompensated or normalized to 35° C., if necessary, in the mannerpreviously described.

The temperature-compensated net reflection intensity values and theknown gray scale reflectance values for the optical standards are thenprocessed using conventional linear regression techniques to define thepredicted relationship between the gray scale reflectance value of anoptical surface and the corresponding reflection intensity value of thesurface measured by the optical reader. The temperature-compensated, netreflection intensity value of the optical reference means 70 is used tosolve the linear regression for the predicted gray scale reflectancevalue of the optical reference means 70. This value is assigned as thegray scale reflectance reference value and is stored for use innormalizing subsequently taken reflection intensity readings of thevarious test sites 84.

In the presently preferred embodiment, the reflection intensity valueread by the optical reader 32 from each test site 84 is converted into acorresponding optical density value. As mentioned previously, theoptical density value of a test site 84 may be directly related to themagnitude of the binding reaction between a capture reagent disposedthereon and a corresponding assay binding component of interest in thetested sample which is specific to the capture reagent. This in turnindicates the degree of allergic sensitivity of the patient to theparticular binding component, where for example the capture reagent is apreselected allergen and the binding component is a human IgE classantibody specific therefor.

The optical density value determined for each test site 84 may berecorded directly as the result of the assay associated with the site.However, it has been found that different IgE class antibodies, eventhough having the same concentrations, produce different levels ofallergic sensitivity in patients brought into contact with allergens forwhich the antibodies are specific. Thus, in a preferred embodiment, theoptical density values are converted to a five level class score rangingfrom 0, which represents no allergic sensitivity, to 4, which representsvery high allergic sensitivity. The five level scoring system allowstest results to be recorded in a uniform format. Each class scorepreferably corresponds to a predetermined range of optical densityvalues which may however be different for different assays. In order toprovide uniformity and to ensure accuracy, the class scores andcorresponding ranges of optical density values for each assay arepreferably statistically correlated to the results of the same allergytests using known techniques such as skin prick and/or RAST testing.

CALIBRATION DATA SYSTEM

In addition to the means described above for providing temperaturecompensation, optical calibration, and conversion of the reflectionintensity values, in a preferred embodiment means are included toprovide assay calibration data for use in calibrating or normalizing theassay results from various test sites on various reaction cartridgeswith respect to common predetermined standard values.

The desirability of providing assay calibration data arises from thefact that the allergens or other assay binding components which arebound to individual test sites during the manufacture of the reactioncartridges are necessarily produced in lots of limited volume. Sinceeach lot cannot be prepared with exactly the same concentration of aparticular binding component as any other lot of the same bindingcomponent, different test sites bound with the same preselected bindingcomponent from different lots can produce different assay results forthe same sample.

Lot-specific assay calibration data provided for each different lotprovides a means for normalizing the assay results associated withindividual test sites on a plurality of reaction cartridges with respectto one or more predetermined common standard values. As a result, lot tolot variations do not appear in assay results because all assay resultsfrom all test sites are normalized to one or more common standards.

Referring to FIG. 14, in a presently preferred embodiment predeterminedassay calibration data 300 is provided, preferably in a machine readableformat, for each lot of assay binding components or capture reagents.The assay calibration data 300 may be provided in any suitable formatand on any suitable data source media, including for example a magneticor punched paper tape format and media. An optical bar code format ispresently preferred and in particular an optical bar code in a formatknown in the art as ASCII 3 of 9. Also in the presently preferredembodiment, the assay calibration data 300 is provided on a paper sheet.

The assay calibration data 300 may include both machine readable andhuman readable information 304 intermixed if desired. The human readableinformation can be quite useful, for example to assist a technician orother operator in determining which lot and panel of assays thecalibration data 300 corresponds to prior to entering the data into theanalyzer 10. A corresponding lot number in human readable form is alsopreferably provided on each reaction cartridge 80 (see FIG. 5) to assistthe operator in selecting the appropriate calibration data 300 for eachlot before initiating a test cycle.

Alternatively, the assay calibration data 300 may be provided in a humanreadable format if manual data entry means such as a keypad areavailable. This is a less preferred alternative because, as will becomeapparent below, a large amount of calibration data must then be enteredmanually which increases the hands-on time and expense associated withthe tests as well as the risk of error.

The machine readable portion of the calibration data preferably includesat least the calibration data 300 and the lot code which the calibrationdata 300 corresponds to. If different panels of assays are available onreaction cartridges 80 manufactured using capture reagents from the samelots, a panel identification code is preferably also included as part ofthe lot code.

It is a significant feature that the calibration data 300 is determinedat the time the reaction cartridges are manufactured so that it isunnecessary for a technician or other operator to manually run standardsor calibrators prior to initiating a test cycle. The calibration data ispreferably generated using a sample from each lot of a capture reagentto assay one or more standard specimens each having a knownconcentration value of a second assay binding component that is specificfor the capture reagent.

As presently preferred, each capture reagent sample is used to assay anumber of standard solutions each having a different known concentrationvalue of a sample assay binding component which is specific for thecapture reagent. The assay results and the known concentrations of theassayed solutions are processed using conventional least-squaresregression techniques to obtain the parameters and type of curve fitthat best describes the calibration curve for each lot of the capturereagent, for example the slope and intercept values for a four pointlinear calibration curve for each lot of the capture reagent. It will beapparent to persons skilled in the art that more or fewer standardsolutions could be assayed depending upon the degree of accuracyrequired or the nature of the calibration curve.

As mentioned above, it should also now be apparent why manual entry ofthe calibration data is not preferred. As an example, given a panel of50 test sites each bound with a different capture reagent, and fivestandard solutions, 250 individual items of calibration data would haveto be manually entered. The number of data items to be entered for agiven test cycle would be further multiplied by the number of reactioncartridges manufactured using capture reagents from different lots.

Preferably, conventional optical code reader means 306 and optical codeprocessing means 308 are provided to enter the calibration data 300 foreach lot from each sheet 302. More specifically, an optical codeprocessing circuit commercially sold by Hewlett-Packard Co. as model no.HBCR-1800 and any commercially available bar code wand that iscompatible therewith may be used. The optical code processing means 308is preferably interfaced to the microprocessor 315 by any suitablemeans, for example a conventional peripheral interface adaptor (PIA).

The microprocessor 315 in turn interfaces in a known manner with a datastorage means 310 which is suitably a conventional RAM memory such asRAM 334 (FIG. 15). In a presently preferred embodiment, themicroprocessor 315 stores the calibration data 300 for each lot in anavailable area 314 of data storage means 310 reserved for such data andstores the starting storage location together with the lot code andpanel code, if any, of the calibration data in a separate lookup table312 either in data storage means 310 or in other storage means ifdesired. As shown in FIG. 14, for example, three sets of calibrationdata, each corresponding to a different lot of capture reagents, areshown stored in data storage means 310 together with the correspondinglot code and starting storage location (in hexadecimal) for each set.

As mentioned previously, code means 94 are preferably provided on eachreaction cartridge 80 delivered to the technician or other operator forcarrying out a panel of assays. In a preferred embodiment, each codemeans includes, among other items of information, a code 318 identifyingthe lot from which the capture reagents bound to the test sites 84 ofthe cartridge originated. In addition, if multiple cartridges containingdifferent preselected panels of assays and manufactured using capturereagents from the same lot are available, the code 318 also preferablyincludes a panel identifying code.

For purposes of this feature of the invention, the code means 94 maytake any suitable format and may be presented on any suitable datasource media. However, it is preferred that the code means 94 be machinereadable and in particular it is preferred that the code means 94 be inan optical bar code format. In addition, although the code means ispreferably applied to the reaction cartridges 80 in the presentlypreferred embodiment, it is understood that the code means could beapplied to other means used for carrying out assays in differentarrangements. Without limitation, other means could include variousfluid containers, reaction containers or cartridges, solid-phase testsubstrates, or the like.

In operation, prior to initiating a test cycle, the operator preferablyinspects the reaction cartridges to be used and notes the lot numbers.The operator then obtains the calibration data sheets 302 having thecorresponding lot numbers and uses the optical code reader means 306 toenter the calibration data from the sheets into the analyzer 10 where itis stored as described above.

During operation of the subsequent test cycle, the analyzer 10preferably uses a second optical code reader 316 to read the code means94 on each reaction cartridge 80 automatically. Suitable optical codereaders are available commercially from numerous sources. Alternatively,the optical reader means 32 could be used to read the code means 94 ifdesired. Less preferably, the code means 94 can be read manually fromeach cartridge 80 using the optical code reader means 306 or othermanually-manipulated code reader means or entered manually using thekeypad.

The microprocessor 315 stores the lot and panel code, if present,entered from each cartridge 80 together with the location of thecartridge 80 on the carousel 18 in a memory 334. At the end of the testcycle when the optical reader 32 reads the assay results from the testsites 84 on each cartridge 80, the microprocessor 315 retrieves the lotcode for each cartridge 80 and compares it with the lot codes previouslystored in the table 312. When a match is found, the microprocessor 315uses the corresponding starting storage location in the table 312 toretrieve the actual calibration data 300 from the storage area 314. Themicroprocessor 315 then uses the calibration data 300 to normalize theassay result for each test site 84, which is determined in the mannerpreviously described, using a regression analysis technique in a mannerwell known to those skilled in the art. Of course, other knownnormalization techniques may be used instead if desired.

SYSTEM CONTROL ARCHITECTURE

Preferably, central control means are provided to control the variousmechanical and electrical elements of the analyzer 10 in a predeterminedmanner to perform various preselected panels of assays simultaneously ona plurality of biological samples. The heart of the central controlmeans is preferably a programmable microprocessor 315 which hasperipheral control, computational, and data processing capabilities. AnIntel 80186 microprocessor has been found to possess the desiredcapabilities and is presently preferred for use as the central controlmeans.

The microprocessor 315 communicates with and controls the variousmechanical and electrical elements of the analyzer 10 by way of itssystem bus 320. The system bus 320 suitably comprises a conventionalcomputer bus having a sufficient number of data, I/O control, andaddress lines to accommodate the preferred microprocessor 315 andinterfaces for the various mechanical and electrical peripheralscomprising the analyzer 10. The selection, interfacing, and operation ofthe system bus is well within the skill of persons of ordinary skill inthe art and further detailed description is unnecessary for a completeunderstanding of the invention.

Program storage means, preferably in the form of PROM 335, is providedto store a control program which contains the instructions necessary forthe microprocessor 315 to control the various electrical and mechanicalelements of analyzer 10 to automatically carry out assays. The writingof such a program is well within the skill of persons skilled in the artgiven the sequence of steps necessary for the microprocessor 315 tocarry out an exemplary panel of assays as set forth in detail below.PROM 335 may be any commercially available PROM compatible with thesystem bus 320 and the preferred microprocessor 315 such as Intel 27512and/or 27010 EPROM's for example.

Additional data storage is preferably provided for system parameterssuch as test site 84 locations relative to the zero position referencecoordinates, assay calibration data, temperature compensation factor,and the like in the form of RAM 334. Suitable RAM is provided by DallasSemiconductor DS1235 RAM's, for example.

Preferably also interfaced to the system bus 320 are keyboard, printer,and display interfaces 322, 324, and 326 which interface a keypad 328,printer 330, and display 332 respectively to the microprocessor 315. Theprinter 330 is preferably a compact thermal printer which may be used toprint out assay results following completion of a test cycle by theanalyzer 10. The printer 330 is suitably any commercially availableprinter which is compatible with the microprocessor 315 and system bus320. The printer interface 324 is preferably a conventional centronicsparallel printer interface connected directly to a DMA channel of thepreferred Intel 80186 microprocessor. Character data to be printed isdownloaded directly from the microprocessor memory to the printerinterface which in turn generates the appropriate control signals tocontrol the printer mechanism.

The keypad 328, which has been described in general terms previously, issuitably a conventional matrix-switch type of keyboard. A conventionalmatrix keypad decoder such as a 74C923 decoder IC preferably decodes thelocation of each depressed key and generates an interrupt to themicroprocessor 315 to communicate the identity of the depressed key forprocessing according to the instructions of the microprocessor controlprogram.

The display 332 is suitably a small LCD type of display which may beused to provide prompts to the operator during a test cycle. The displaymay be interfaced to the microprocessor 315 in conventional fashionusing a display driver, such as a commercially available Hitachi HD44100driver IC, and a display controller, such as a commercially availableHitachi HD44780 controller IC. Character data to be displayed istransmitted by the microprocessor 315 to the display controller which inturn controls the display driver to generate the appropriate displaysignals to display the character data.

In a particularly preferred embodiment, all of the program storage,additional storage, microprocessor, and keypad, printer, and displayinterface means are provided on a single CPU board which is commerciallyavailable from Intel Corp. of Santa Clara, Calif.

The stepper motor drives for the carousel 18 and boom arm 30 are eachinterfaced to the microprocessor 315 by way of an interface 338. Eachinterface 338 preferably comprises a programmable interface timer (PIT)such as an 8254 type PIT and a programmable logic controller (PLC) suchas a GAL16V8 type PLC. In order to cause a stepper motor to move anumber of steps, the microprocessor 315 preferably programs the PIT tocount down a selected number of counts at a selected frequency. The PLCis responsive to the PIT counts to output the programmed number of steppulses to the stepper motor at the programmed frequency.

The wash and waste pumps are interfaced to the microprocessor 315through a wash/waste pump control interface 352 which preferablycomprises a pair of conventional motor control relays. Themicroprocessor 315 outputs motor on and off signals to open and closethe relays directly and thereby apply power to and remove power from thepump motors.

The temperature sensors and heaters mentioned previously for controllingthe temperature in the processing chamber 11 are interfaced to themicroprocessor 315 through temperature sensor and heater on/off controlinterfaces 346 and 348. The temperature sensor interfaces 346 preferablycomprise A-D converters, most preferably of the voltage to frequencytype, and counters arranged and operated in the same manner as describedabove with respect to the preferred optical reader signal processing andcontrol circuit 225. In order to read a sensor, the microprocessor 315programs a PIT 344 to provide an integration period for the counter andenables the counter. When the PIT signals that the programmedintegration interval is over, the microprocessor 315 disables thecounter, reads the final count value which represents the measuredtemperature, and resets the counter for the next read.

The heater on/off control interface preferably comprises a heater on/offcontrol relay. Power to the heater is controlled directly by themicroprocessor 315 transmitting logic signals to the interface to openand close the relay.

The optical reader signal processing and control circuit 225 andassociated DAC 342, the boom arm and carousel optical switches 350, andthe optical code reader means 306 and interface 308 have all beendescribed in detail previously in conjunction with the microprocessor315.

EXEMPLARY MODE OF OPERATION

A detailed step by step description will now be given of a preferredmode of operation of the presently preferred biological sample analyzer10 in carrying out an exemplary panel of enzyme immuno assays (EIA's) oneach of a plurality of biological samples.

When power is first applied to the analyzer 10, the microprocessorcontrol program preferably causes the microprocessor 315 to go through aseries of steps preparatory to performing a test cycle. Initially, themicroprocessor 315 programs the drive interfaces 338 of the boom arm 30,the probe arm 33 and carousel 18 stepper motors 50, 56 and 66 toposition the boom arm 30, probe arm 33 and carousel 18 in theirrespective home positions. The microprocessor 315 then awaitsacknowledgement, in the form of signals from the optical switches 350associated with the boom arm 30, probe arm 33 and carousel 18, that theyhave reached their respective home positions.

After the boom arm 30 and carousel are homed, the microprocessor 315programs the boom arm and carousel drive interfaces 338 to cause thestepper motors 50 and 56 to rotate the boom arm 30 and carousel 18 intopositions where the optical reader 32 is adjacent to the opticalpositioning means 140. The microprocessor 315 next sends an LED CONTROLsignal to the optical reader processing and control circuit 225 to turnon the LED 162. The microprocessor 315 then sequentially programs thedrive interfaces 338 for the boom arm 30 and carousel 18 to cause theoptical reader 32 to first scan the parallelepiped structure of theoptical positioning means 140 radially while the carousel 18 remainsstationary, and then for the carousel 18 to rotate the parallelepipedstructure past the stationary optical reader 32 circumferentially.During the scanning process, the microprocessor 315 initiates aplurality of equally spaced optical readings in the manner previouslydescribed and stores each reflection intensity reading in RAM 334. Fromthe stored readings, the microprocessor 315 calculates the coordinatesof the center of the paralellepiped structure as the number of countsfrom home for the carousel and boom arm stepper motors 50 and 56 asdescribed previously and stores the coordinates as the zero-positionreference. The microprocessor 315 then waits for the initiation of atest cycle.

All subsequent positioning of the carousel 18 and boom arm 30 to accessany particular location on the carousel 18 or a cartridge 80 ispreferably accomplished by the microprocessor 315 by programming thedrive interfaces 338 with a number of predetermined carousel and boomarm stepper motor steps which correspond to the location of interest.These steps are preferably predetermined and stored in a location tablein RAM 334. The location table (not shown) preferably includespredetermined stepper motor count values for positioning the carouseland boom arm at predetermined positions to provide optical access to thecode means 94, probe 28 access to the ports 92 of the reaction wells 86in a fluid access position, and optical access to each of the test sites84 in a reaction well 86 of a cartridge 80 in a reading position.Preferably, the step values stored in the location table are takenrelative to the coordinates of the stored zero position reference. Thus,the actual number of steps for the boom arm or carousel to reach aparticular location from the home position is the sum of the zeroposition reference coordinates and the step values in the locationtable.

Prior to initiating a test cycle, the operator obtains the appropriatereaction cartridges 80 for the tests to be performed and notes the lotnumbers. The operator then obtains the assay calibration data sheet 302for each lot number and enters the calibration data 300 for each lotusing the optical code reader means 306. The microprocessor 315 respondsto the optical code reader means 306 in accordance with the controlprogram to store the calibration data 300 and the lot number andstarting storage location data pair in RAM 334 as described above.

The operator preferably initiates a test procedure by pressing the RUNkey on the keypad 34. In accordance with the control program, themicroprocessor 315 responds to the RUN key at this point by programmingthe drive interfaces 338 to home the boom arm 30 and carousel 18. Themicroprocessor 315 then transmits a character string to the displayinterface 326 to prompt the operator on the display 36 to place areaction cartridge in the carousel opening at the front of the analyzer10 and enter the patient ID.

In the exemplary enzyme immuno assay being described, the operatorpreferably introduces approximately 0.5 ml of patient serum and 0.5 mlof a specimen dilution buffer, such as 10% heat inactivated horse serumin 10 mM (TB5) pH 7.4, into the reaction well 86 of a cartridge 80through the port 92. The operator then preferably manually records apatient identification code on the reaction cartridge 80, loads thecartridge 80 into the opening in the carousel 18 and enters the patientidentification code on the keypad 34. The microprocessor 315 responds tothe entry of the patient identification code on the keypad 34 inaccordance with the control program by storing the location of thecartridge on the carousel 18 with the patient identification code in RAM334.

The microprocessor 315 then waits for the next entry from the keypad 34.The operator preferably depresses either the INDEX key or the RUN key.If the operator depresses the INDEX key, the microprocessor 315 respondsby programming the carousel drive interface 338 to cause the carouselstepper motor 56 to rotate the carousel 18 one cartridge position andpresent the next carousel opening at the front of the analyzer 10. Thisprocess is repeated until all cartridges containing samples to be testedhave been loaded on the carousel. When, the operator depresses the RUNkey, the microprocessor 315 responds in accordance with the controlprogram to initiate execution of the appropriate test procedures for thesamples.

The microprocessor 315 first programs the carousel and boom arm driveinterfaces 338 with the step coordinates to sequentially position theoptical reader 32 adjacent to the code means 94 on each cartridge 80 andthen to scan each code means 94. During each scan, the microprocessor315 operates the optical reader 32 to take a plurality of reflectionintensity readings of the code means 94 and stores the readings.Following each scan, the microprocessor 315 processes the storedreadings and derives the code means 94 from the contrast betweenreflection intensity readings taken from light and dark areas of thecode. Alternatively, as mentioned previously, the microprocessor 315 maycontrol other conventional optical code reader means to read the codemeans 94 if desired.

The microprocessor 315 then processes the code means 94 and derives anassay type code therefrom. The microprocessor 315 is preferablyresponsive to the assay type code in accordance with the control programto subsequently carry out the steps necessary to perform the identifiedassay within the parameters of a predetermined protocol for the assay.For purposes of the present description, it is assumed that an enzymeimmuno assay is to be performed using a sandwich assay format.

The microprocessor also derives the lot code from the code means 94 andstores it in RAM 334 until needed, together with the correspondingpatient identification code and carousel position data.

After the code means 94 on each reaction cartridge 80 has been read, themicroprocessor 315 in accordance with the assay type code and thecontrol program initiates a timed incubation cycle. In the exemplary EIAbeing described, the microprocessor 315 programs one or more of thePIT's 344 to time a sample incubation cycle of 16 hours. During theincubation cycle, the microprocessor 315 programs the carousel driveinterface 338 to continuously rotate the carousel to provide gentleagitation and promote binding of the IgE class antibodies in each samplewhich are specific for the capture allergens bound to the test sites 84of each reaction cartridge 80.

When the incubation cycle times out, the microprocessor 315 preferablyinitiates a wash procedure in accordance with the assay type code andthe control program. The microprocessor 315 programs the boom arm andcarousel drive interfaces 338 to position the boom arm in apredetermined fluid access position and to sequentially position eachcartridge on the carousel under the probe 28 at the fluid accessposition. As each cartridge is brought under the probe, themicroprocessor 315 programs the drive interface 338 for the linearstepper motor 66 to pivot the probe 28 downwardly through the port 92and into the reaction well 86. The microprocessor 315 then transmitssignals to the wash/waste pump control interface 352 to sequentiallycause the waste pump 27 to aspirate the serum sample from the reactionwell 86, the wash pump 25 to introduce a volume of wash fluid into thereaction well 86, the carousel 18 to rotate for a predetermined time,and the waste pump 27 to aspirate the spent wash fluid from the well 86.The microprocessor preferably repeats the steps of introducing, rotatingand aspirating wash fluid about three times. The microprocessor thenprograms the drive interface 338 to cause the linear stepper motor 66 topivot the probe 28 upwardly out of the reaction well. When the reactionwells 86 of all cartridges 80 have been washed, the microprocessorprograms the drive interface 338 to home the boom arm.

The microprocessor 315 next transmits a prompt string to the displayinterface 326 to prompt the operator to introduce conjugate reagent tothe reaction cartridges 80. In the exemplary EIA being described, theanalytes or sample binding components of interest in the samples arehuman IgE class antibodies and the conjugate is preferably goatimmunoglobulin which is specific for the epsilon chain of human IgEclass antibody conjugated to an enzyme such as alkaline phosphatase orhorse radish peroxide (HRPO) conjugate. However as is known in the art,the specific detecting conjugate employed may be varied as long as adetectable label (enzymatic or fluorogenic, for example) is linked to aspecies (antibody or other identifiable binding agent, for example)capable of detecting the analyte of interest or the analyte-capturereagent complex. It is conceivable to utilize coloidal conjugates suchas gold or other metal as the detectable label.

The operator introduces the conjugate to the reaction well 86 of thecartridge 80 at the front of the carousel, preferably through the port16 in the processing chamber door 12, then depresses the INDEX key. Themicroprocessor 315 responds by programming the drive interface 338 toindex the carousel 18 by one position. This procedure repeats until theoperator has introduced conjugate to each reaction cartridge 80.

When the conjugate introduction procedure is completed, themicroprocessor 315, in accordance with the assay type code and thecontrol program, initiates another timed incubation cycle in the samemanner as described above. In the case of the exemplary EIA beingdescribed, the conjugate incubation period is preferably approximatelyfour (4) hours. During the conjugate incubation period, themicroprocessor 315 also causes the carousel stepper motor 56 tocontinuously rotate the carousel 18 to provide gentle agitation andpromote binding of the conjugate to the antibody capture reagent-testcard complex.

When the conjugate incubation cycle times out, the microprocessorinitiates a second wash procedure in substantially the same manner asthe first wash procedure. Following the wash procedure, themicroprocessor causes the operator to be prompted to introduce asubstrate reagent. In the case of the exemplary EIA being described, foran HRPO enzyme label, a preferred substrate is 4-chloro-1-naphthol inisopropanal and hydrogen peroxide. For an alkaline phosphate label, apreferred substrate is 5-bromo-4-chloro-3 indolyl phosphate/Nitro bluetetrazolium (BCIP/NTT) in aminomethyl-propanal. In both cases, thesubstrate is selected to be acted upon by the enzymatic label of theconjugate to develop a color on the surface of each test site 84 havingbound the specific analyte of interest. The colored test site has anoptical density related to the magnitude of the binding reaction betweenthe allergen bound to the test site and the sample analyte specific forthe allergen. The optical density can be determined from the reflectionintensity of the test site read by the optical reader 32 to obtain thedegree of allergic sensitivity of the patient to the capture allergen.

The microprocessor preferably operates in accordance with the controlprogram to index the carousel 18 and prompt for introduction of thesubstrate into each reaction cartridge 80 in the same manner asdescribed above with respect to the conjugate. Following the substrateintroduction procedure, in the exemplary EIA, the microprocessorinitiates a timed substrate incubation cycle during which the substrateis allowed to remain in contact with the test sites 84 of each reactioncartridge for approximately thirty minutes. During this period, themicroprocessor causes the carousel to rotate continuously to providegentle agitation in the same manner as described above.

Following the substrate incubation period and wash, the microprocessor,operating in accordance with the control program, initiates a dryingprocedure. Initially, the microprocessor causes the carousel 18 to berotated to its home position. The microprocessor then causes the displayto prompt the operator to remove the cover 90 from the reactioncartridge 80 in the front position of the carousel. The operatorpreferably opens the door 12 of the analyzer 10 and removes each cover90 as prompted by peeling it off the top of the reaction well wall 88.The cover 90 may then be discarded. The microprocessor waits for theoperator to depress the INDEX key. When the operator depresses the INDEXkey, the microprocessor causes the carousel to be rotated by one openingso that the next cartridge 80 is presented at the front position. Themicroprocessor causes the display to again prompt the operator to removethe cover 92 of the cartridge in the front position. This procedurerepeats until the operator removes the covers from all of the cartridges80 on the carousel 18.

When the cover is removed from the last cartridge and the operatordepresses the RUN key, the microprocessor causes the display to promptthe operator to close the processing chamber door 12 and depress the RUNkey again. When the operator depresses the RUN key, the microprocessorresponds in accordance with the control program to program a timeddrying interval of preferably approximately fifteen (15) minutes and tocause the carousel stepper motor 56 to rotate the carousel continuouslyduring the timed interval to promote drying of the test cards 82 in eachof the cartridges 80. Also at this time, the dark reflection intensityis first determined with the LED off; then the LED is energized andwarms up during the drying cycle.

When the drying interval times out, the microprocessor, in accordancewith the control program, automatically initiates a reading procedure,beginning with a reading of the optical reference means 70. Themicroprocessor sequentially programs the carousel and boom arm driverinterfaces 338 to sequentially position each reaction cartridge 80 onthe carousel 18 in a predetermined reading position which intersects thearcuate path of motion of the optical reader 32. Once a reactioncartridge 80 is in the reading position, the microprocessor sequentiallyretrieves the step coordinates for each test site 84 from the locationtable and causes the carousel and boom arm stepper motors tosequentially position the carousel and boom arm to read each test site84 in the manner described previously in detail. The microprocessor netsthe reflection intensity reading for each test site, temperaturecompensates it if necessary, and stores it in RAM 334 with the carouselposition and patient identification code of the cartridge 80.

When each test site 84 of each reaction cartridge 80 has been read, themicroprocessor retrieves the readings for each cartridge 80 from RAM,converts them to an optical density, calibrates them using the storedassay calibration data, and then converts them to a class score all inthe manner described previously, and stores them back in DMA accessiblememory. When all of the readings have been calibrated and converted, themicroprocessor initiates a DMA transfer of the stored test results tothe printer interface 324 which in turn causes the printer to print thetest results for each patient identification code in the form ofnormalized class scores for each capture allergen of the panel ofassays.

After the test results are finished printing, the microprocessortransmits a prompt string to the display interface 326 to prompt theoperator to remove the used cartridges 80 from the carousel 18. Themicroprocessor controls the indexing of the carousel and the removal ofthe spent cartridges in substantially the same manner as theintroduction of the various reagents described above.

The foregoing description of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. It is not intended to limit the scope of the invention,which is defined by appended claims and their equivalents. Variousmodifications and variations of the preferred embodiments are possiblein light of the above teachings and will be apparent to persons skilledin the art. Such modifications and variations do not depart from thespirit or scope of the invention and it is therefore intended that thescope of the invention be defined by the following claims, including allequivalents.

We claim:
 1. Apparatus for providing assay information useful forcalibration or normalization of data obtained from a panel of assays fora plurality of selected test sample binding components performed on abiological sample, comprising:predetermined assay calibration data forcalibrating or normalizing the results of a preselected panel of assaysfor a plurality of selected test sample binding components with respectto at least one predetermined standard value for each said bindingcomponent; data providing means for providing said predetermined assaycalibration data, wherein said data providing means containspredetermined assay calibration data for said panel of assays and firstcode means for identifying said predetermined assay calibration data ofsaid preselected panel of assays; storage means for storing saidcalibration data and said first code means; entering means for enteringsaid calibration data and said first code means from said data providingmeans to a location in said storage means without performing an assay;assay means for simultaneously carrying out a preselected panel ofassays on a single aliquot of a biological sample, wherein said assaymeans contains a preselected panel of assay reagents wherein each assayreagent is bound to its own discrete test site in an array of isolatedtest sites and second code means for identifying said panel of assays;and correlation means for correlating assay results for said panel ofassays of said assay means with said predetermined assay calibrationdata for said panel of assays in said storage means using said firstcode means and said second code means.
 2. The apparatus defined in claim1 wherein:said predetermined assay calibration data and said first codemeans of said data providing means is provided in a machine-readableformat; and said entering means includes reader means for reading saidmachine-readable calibration data and first code means.
 3. The apparatusdefined in claim 1 including a plurality of said data providing meansfor providing said predetermined assay calibration data, where each saiddata providing means contains predetermined assay calibration data for adifferent preselected panel of assays and a different first code meansfor identifying said predetermined assay calibration data of saidpreselected panel of assays.
 4. The apparatus defined in claim 1 whereinsaid assay means comprises disposable reaction cartridge means havingbound thereto said array of isolated test sites a plurality ofpreselected capture reagents for a preselected panel of assays.
 5. Theapparatus defined in claim 1 wherein:said second code means of saidassay means is provided in a machine-readable format; and saidcorrelation means includes reader means for reading saidmachine-readable second code means.
 6. The apparatus defined in claim 1including:controlling means for controlling said entering means to causesaid entering means to enter said predetermined assay calibration dataand first code means in a selected available storage location of saidstorage means; first associating means for associating said first codemeans and said predetermined assay calibration data with said selectedavailable storage location of said storage means; and second associatingmeans for associating said second code means with said selectedavailable storage location of said storage means for accessing saidcalibration data stored in said location.
 7. Apparatus for providingassay information useful for calibration or normalization of dataobtained from panels of assays for a plurality of selected test samplebinding components performed on a plurality of biological samples,comprising:predetermined machine-readable assay calibration data forcalibrating or normalizing the results of preselected panels of assaysfor a plurality of selected test sample binding components with respectto at least one predetermined standard value; data providing means forproviding said predetermined machine-readable assay calibration data,wherein said data providing means contains said predeterminedmachine-readable assay calibration data for said panels of assays andfirst code means for identifying said predetermined assay calibrationdata of said preselected panels of assays; storage means for storingsaid calibration data for a plurality of said preselected panels ofassays and said first code means; user-operable reader means for readingsaid calibration data and said first code means from said data providingmeans; entering means for entering said calibration data and said firstcode means read by said user-operable reader means in selected availablestorage locations in said storage means without performing an assay;associating means for associating said predetermined assay calibrationdata and said first code means read from said data providing means withsaid selected available storage location of said storage means; aplurality of disposable reaction cartridge means each designed forsimultaneously carrying out a preselected panel of assays on a singlealiquot of a biological sample wherein each said reaction cartridgemeans contains a preselected panel of assay reagents wherein each assayreagent is bound to its own discrete test site in an array of isolatedtest sites, and machine-readable second code means for identifying saidpanel of assays; reader means for reading said second code means fromeach said reaction cartridge means; associating means for associatingsaid second code means with said selected available storage location ofsaid storage means for accessing said calibration data stored in saidlocation; and correlation means for correlating assay results for saidpanel of assays of each said reaction cartridge means with saidpredetermined assay calibration data for each said panel of assays insaid storage means using said first code means and said second codemeans.
 8. A method for providing assay calibration data to an instrumentcapable of simultaneously assaying a single aliquot of a biologicalsample with a panel of assays of preselected test sample binding,components comprising the steps of:providing predetermined assaycalibration data for calibrating or normalizing the results of saidpanel of assays with respect to at least one predetermined standardvalue for each said binding component and first code means foridentifying said calibration data; entering said calibration data into alocation in a data storage means of said instrument; providing assaymeans for carrying out said panel of assays with said instrument,wherein said assay means comprises an array of isolated test siteswherein each assay reagent is bound to its own discrete test site, andsecond code means for identifying said panel of assays; and accessingsaid calibration data in said data storage means using said first codemeans and said second code means.
 9. The method defined in claim 8wherein:said predetermined assay calibration data is provided in amachine-readable format; and said predetermined assay calibration datais entered into said data storage means with a reader means for readingsaid machine-readable predetermined assay calibration data.
 10. Themethod defined in claim 8 wherein said assay means comprises disposablereaction cartridge means having bound to said array of isolated testsites a plurality of preselected capture reagents for carrying out apreselected panel of assays with said instrument.
 11. The method definedin claim 8 wherein:said second code means is machine-readable; and thestep of accessing said calibration data in said data storage meansincludes reading said machine-readable second code means.
 12. The methoddefined in claim 8 including the steps of:associating said first codemeans and said location in said storage means, where said location insaid storage means is a selected available storage location, andaccessing said calibration data in said storage means using saidselected available storage location associated with said first codemeans and said second code means.